34

CHAPTER - 2

SIMULTANEOUS DETERMINATION OF ORLISTAT AND SIBUTRAMINE

HYDROCHLORIDE RELATED SUBSTANCES IN APSULES

FORMULATION AND IDENTIFICATION AND CHARACTERIZATION OF

IMPURITIES

2.1 OBJECTIVE:

To develop simultaneous determination method of orlistat and

sibutramine hydrochloride related substances in capsules Formulation,

Identification of impurities and Characterization of sibutramine

hydrochloride impurities.

2.2 INTRODUCTION:

Orlistat [135] is known as tetrahydrolipstatin, a drug prepared to

treat obesity [136]. It prevents absorption of fats from the human diet

and it is primary function, thereby reducing caloric intake. It is preferred

to use with a physician-supervised reduced calorie diet. Orlistat is a

potent natural inhibitor of pancreatic lipases isolated from the bacterium

streptomyces toxytricini and the saturated by-product of lipstatin [137].

However, due to stabilisation, orlistat rather than lipstatin was developed

as an anti-obesity drug [138]. The primary route of removal is through

the feces. It obstructs approximately 30% of dietary fat from being

absorbed [139] by over-the-counter dose of 60 mg [140, 141]. Higher

doses do not produce more strong effects [142]. Weight loss may increase

the risk of orthopedic problems and mineral loss. Therefore, inhibition of

35

dietary fat during absorption and result to inhibit intestinal lipase

activity during orlistat treatment could have a severe effect on dietary

mineral absorption and balance due to formation of insoluble mineral

soaps within the intestine [143].

Sibutramine hydrochloride monohydrate administered for the

treatment of obesity, as a hunger suppressant. It is a serotonin-

norepinephrine centrally-acting reuptake inhibitor chemical orally

structure related to amphetamine [144], although its mechanism of

action is clear [145- 148].

2.3 LITERATURE REVIEW:

Bennett P. et al. were reported a rapid, sensitive and specific

analytical method for orlistat determination in human plasma. In this

method reconstituted extracts of orlistat were analyzed by HPLC carried

out by using a 50 x 2mm i.d., 3 μm size of the particle, phenyl column.

The mobile phase used was acetonitrile and 2 mM ammonium acetate in

the ratio of 90:10, ion spray tandem mass spectrometry detector was

used for detection. The calibration graphs were found linear in the range

0.20 to 10 ng/mL [149].

Wieboldt R. et al. were reported the use of a quadrupolar ion trap for

quantitation in a bioanalytical laboratory. The orlistst evaluation was

completeded with the cross-validation of an LC-MS-MS quantitative

method first validated on a triple quadrupole mass spectrometer [150].

36

Mohammadi A. et al. were stated a stability-indicating high performance

liquid chromatographic method for the determination of orlistat in

capsules. In this method an isocratic separation was got by using a

Perfectsil ODS-3, 250 mm × 4.6 mm i.d., 5 μm particle size columns with

a flow rate of 0.7 ml/min by using a UV detector to monitor at 210 nm.

The mobile phase contains mixture of

methanol:acetonitrile:trifluoroacetic acid in the ratio of 82.5:17.5:0.01,

v/v/v respectively. Separation was achieved for all degradation products

and parent compound in an overall analytical run time of approximately

15 min [151].

Hassan K. A. et al were reported a HPLC method with UV detection for

determination of orlistat, a Nova- Pack C18 column and an isocratic

mobile phase of 0.1% phosphoric acid solution and acetonitrile in the

ratio of 10:90, v/v used respectively. Detection was carried out at 205

nm. Orlistat was eluted at 6 min without interfering peaks from placebo.

The method was found linear over the range of 10-160-μg/ml orlistat

[152].

Hakala K.S. et al were reported liquid chromatography with ion trap

mass spectrometry method used for the identifition and structural

characterization of by-products of the anti-obesity drug sibutramine.

Metabolites and their isomers formed via dehydrogenation,

hydroxylation, and demethylation, addition of CO2, acetylation and

glucuronidation were observed in MS experiments. However, evident

37

enantioselective formation was identified for two hydroxyl by-products

and two glucuronide conjugates, showing that the hydroxyl/glucuronic

acid moiety in those structures [153].

Radhakrishna T. et al were reported two isocratic liquid

chromatography methods for the purity standardisation and quantitative

estimation of sibutramine HCl, using 4-chloro aniline and lovastatin as

internal standards, respectively. The ratio of variances was close to unity,

correlation coefficient was more than 0.999. The accuracy estimated as

relative mean error for the intra-day assay is ±1.7%. The enantiomeric

separation of sibutramine by chiral chromatography method was

elaborated and this method was shown capable of partetioning the two

enantiomers with a selection of 1.4 and a resolution of 4.0. These two

methods are most useful in the quality control and found to be stability

indicaters [154].

Segall A.I. et al were reported the estimation of sibutramine

hydrochloride in the presence of its oxidatively-induced degradation

products by reversed-phase HPLC. The above method was validated with

respect to stability-indicating method parameters by forced degradation.

The chromatographic conditions includes C18 column and mobile phase

used was mixture of methanol, water, triethylamine (80:20:0.3) and the

pH was adjusted to 4.5 with 85% phosphoric acid, and operated at the

flow rate of 1.1 mL per minute. The eluent was monitored at 225 nm

[155].

38

Singh A.K. et al were reported UV-derivative spectrophotometry and

HPLC methods and compared for the estimation of sibutramine in drug

products. The UV-derivative spectrophotometry and HPLC methods were

found to be statistically exact and precise, there was no variation

observed between the proposed UV-derivative spectrophotometry and

HPLC methods. An alpha-1-acid glycoprotein column used for the

enantiomeric separation of sibutramine. The R- and S-sibutramine were

eluted in less than 5 min with baseline separation of the

chromatographic peaks (alpha = 1.9 and resolution = 1.9) [156].

Chen J. et al were reported a sensitive and selective method for the

estimation of the active primary amine metabolite of sibutramine, N- di-

desmethylsibutramine in human plasma, based on HPLC–MS–MS. ODS

column was used for chromatographic separation using a mobile phase

contains acetonitrile–0.1% trifluoroacetic acid (55:45, v/v) at a flow- rate

of 0.3 ml/min. Multiple reaction monitoring using precursor to product

ion combinations at m/z 252 [157].

Chandorkar J.G. et al were reported a rapid reproducible and specific

reverse phase HPLC method for the determination of Sibutramine and it‘s

impurities in Bulk as well as formulation. The analyte was separated by

using an ODS, C8, RP Column, 4.6 x 250 mm, 5 μm, mobile phase used

was sodium dihydrogen phosphate and acetonitrile at the flow rate of 1.0

mL/min, at wavelength of 230 nm . HPLC system used was Jasco

39

make, UV visible detector of model UV 1575 & Jasco make HPLC pump

of model PU 1580 [158].

Katarzyna M.S. et al were reported quality studies of

sibutramine using HPLC-ED, HPLC-MS/MS and X- ray powder

diffraction (XRPD) methods. Especially the XRPD assisted with an optical

microscopy seems to be useful as a fast screening method of general

sample composition of such preparations. First of all it can discriminate

between capsules containing pure herbal materials and those with some

chemical additives. In the case of mixtures of different chemical additives

further studies such as HPLC-ED and HPLC-MS/MS are helpful and

experience proved that the most often used additive in different herbal

preparations is sibutramine [159].

Young U.S. et al were reported a simple HPLC method for estimation

of active by-products of sibutramine, N- mono-desmethyl metabolite and

N-di-desmethyl metabolite in the rat administred serum sibutramine

HCl. Rat serum was injected directly onto the precolumn. Mobile phase

employed was mixture of methanol, acetonitrile, 20 mM ammonium

phosphate buffer pH 6.0 with phosphoric acid in the ratio 8.3:4.5:87.2

v/v/v and detected at 223 nm [160].

Ding L. et al were reported a sensitive LC-MS method for

simultaneous estimation of sibutramine and its by-products by HPLC on

a reversed phase C18 column with a mobile phase comprising of 10 mM

40

ammonium acetate buffer adjusted to pH 3.5 with acetic acid and

methanol in the ratio 25:75 v/v. These analytes were determined by

using electrospray ionization in a single quadrupole mass spectrometer.

Selected-ion monitoring mode to target ions at m/z: 280 for sibutramine

[161].

2.4 THEORETICAL ANALYSIS

2.4.1 Sample Information [162]

Orlistat is a molecule having the chemical name as [(1S)-1-[(2S,3S)-3-

hexyl-4-oxo-oxetan-2-yl]methyl] dodecyl] (2S)-2-formamido-4-methyl-

pentanoate, molecular formulae of C29H53NO5 and reported molecular

weight 495.735 g/mol. Orlistat is practically insoluble in water, freely

soluble in ethanol (96%) and acetonitrile organic polar solvents, organic

non-polar solvents, and in water. Orlistat is non-ionic basic compound

contains chiral centers and with a negative optical rotation in ethanol at

529 nm and a single diastereomeric molecule. Orlistat pKa and log P are

6.5 and 7.61 respectively.

Fig. 2.01

Orlistat Structural Formula

41

Sibutramine [163] is a molecule having the chemical name as 1-[1-(4-

chlorophenyl) cyclobutyl]- N,N,3- trimethylbutan- 1-amine and having a

molecular formulae of C17H26ClN and reported molecular weight 279.85

g/mol. Sibutramine hydrochloride is soluble in acetone, acetonitrile and

sparingly soluble in methanol. Sibutramine hydrochloride is ionic basic

compound. The pH, pKa and log p values for Sibutramine hydrochloride

are 8.5, 5.2 and 5.05 respectively.

42

Fig. 2.02

Sibutramine Hydrochloride Monohydrate Structural Formula

Sample selected was in solid state and complex mixture of

sibutramine hydrochloride, orlistat and other excipients. Both active

ingredients are in capsules formulation and which are soluble in

acetonitrile and other excipients are not soluble in acetonitrile, so

acetonitrile and water combination as a solvent can be selected for

extraction of these drugs from capsule formulation. Sibutramine HCl and

orlistat are having UV absorption.

In summary, reverse phase chromatographic separation is suitable for

method development. Columns suitable for sibutramine hydrochloride

and orlistst compounds in reversed phase chromatography are C18, C8,

phenyl, and cyano.

43

Table 2.01

Initial HPLC Method Development conditions for Orlistat and

Sibutramine HCl

Separation Variable Preferred Initial choice

Column

Dimensions(length,ID) 15x0.46 cm

Particle size 5 μma

Stationary phase C8 or C18

Mobile Phase

Solvents A and B Buffer-acetonitrile

% B 80-100%b

Buffer(compound, pH,

concentration)

25 mM potassium

phosphate, 2.0<pH<3.0c

Additives(e.g.,amine modifiers, ion-pair reagents)

Not recommended initially

Flow rate 1.0-2.0 ml/min

Temperature 25 °C

Sample size 20 μL

2.5 EXPERIMENTAL INVESTIGATIONS

44

2.5.1 Experiment No. 1

Potassium dihydrogen phosphate GR grade, Orthophosphoric acid GR

grade, Acetonitrile HPLC grade, Water milli Q, Sibutramine hydrochloride

WS and Orlistat WS chemicals and reagents were used for experiments.

The reference of sibutramine hydrochloride was kindly supplied by

Hetero Drugs Limited (Hyderabad, India) and capsules® was obtained in-

house sample. Capsules are claimed to contain 10 mg of sibutramine

hydrochloride, 120 mg of orlistat and following inactive ingredients. All

reagents were analytical or HPLC grade. Acetonitrile and methanol were

procured from Mecrk (Mumbai, India), the phosphoric acid from Merck

(Mumbai, India). Purified water was obtained by a Millipore® Dire-Q 3 UV

with pump (Molsheim, France)

The HPLC system (Agilent 1200 series, Santa Clara, USA) consisted of

a G1311A quaternary pump, G1322 vacuum degasser, G1316A

thermostat column compartment, G1329A standard auto sampler and

G1315B diode array detector set at 210 nm. The mobile phase was

acetonitrile and a solution of 50 mM potassium dihydrogen phosphate

buffer adjusted pH to 3.0 with 10% solution of phosphoric acid, (60:40;

v/v). Mobile phase was filtered through 0.45 μ membrane filter. The

analytical column, an inertsil ODS-3V, 250 mm x 4.6 mm, 5 µ, was

operated in ambient temperature (25° C). Washed the column with milli-

45

Q water and methanol for few minutes then saturated the column with

the mobile phase for 30-40 min.

Mobile phase flow rate was seted at 1.5 ml/min. Standard solution for

Sibutramine hydrochloride and orlistat solution was prepared having

concentration of 1200 μg/mL of Orlistat and 100 μg/mL of Sibutramine

respectively in mobile phase. Injected 20 μL standard solutions two times

and average detector response measured at 210 nm. Chromatograms

evaluated with respect to retention time, resolution and peak shape.

Only one peak was eluted at 5 min and second component peak was

not eluted upto 40 min. The eluted peak was confirmed as sibutramine

hydrochloride. Orlistat peak was not eluted in these chromatographic

conditions.

2.5.2 Experiment No.2

The mobile phase was acetonitrile and a solution of 50 mM

potassium dihydrogen phosphate buffer adjusted pH to 3.0 with 10%

solution of phosphoric acid, (90:10; v/v). Mobile phase was filtered

through 0.45 μ membrane filter. The analytical column, inertsil ODS-3V,

250 mm x 4.6 mm, 5 µ, maintained at 25 °C temperature. The mobile

phase flow rate was maintained at 1.5 ml/min. Standard Sibutramine

hydrochloride and orlistat solution was prepared at concentration, 1200

μg/mL of Orlistat and100 μg/mL of Sibutramine hydrochloride in mobile

phase. 20 μL standard solutions were injected two times and average

46

detector response taken at 210 nm. Chromatograms evaluated according

to retention time, resolution and peak shape.

Sibutramine eluted at 1.5 minutes and orlistat which were not eluted

up to 30 minutes and the next experiment carried out with methanol and

buffer as mobile phase.

2.5.3 Experiment No.3

The mobile phase was acetonitrile and a solution of 50 mM potassium

dihydrogen phosphate buffer set at pH to 3.0 with 10% solution of

phosphoric acid, (90:10; v/v). Mobile phase was filtered through 0.45 μ

membrane filter. The analytical column, inertsil ODS-3V, 250 mm x 4.6

mm, 5 µ, maintained at temperature 25 °C. The mobile phase flow rate

was maintained at 1.5 ml/min. Standard Sibutramine hydrochloride and

orlistat solution was prepared at concentration, 1200 μg/mL of orlistat

and 100 μg/mL of sibutramine hydrochloride in mobile phase. 20 μL

standard solutions were injected two times and average detector

response measured at 210 nm. Chromatograms evaluated with respect to

retention time, resolution and peak shape.

Sibutramine eluted at 1.5 minutes orlistat which was not eluted up

to 50 minutes. Hence it is concluded that the acetonitrile is favorable for

elution of orlistat rather than methanol, gradient elution required or

column polarity can be decreased.

47

2.5.4 Experiment No.4

The mobile phase was acetonitrile and a solution of 50 mM potassium

dihydrogen phosphate buffer adjusted pH to 3.0 with 10% solution of

phosphoric acid, (50:50; v/v). Mobile phase was filtered through 0.45 μ

membrane filter. The analytical column, Zorbax SB phenyl, 250 mm x

4.6 mm, 5 µ, maintained at temperature 25 °C. The mobile phase flow

rate was maintained at 1.5 mL/min. Standard Sibutramine

hydrochloride and orlistat solution was prepared at concentration, 1200

μg/mL of orlistat and 100 μg/mL of sibutramine hydrochloride in mobile

phase. 20 μL standard solutions were injected two times and average

detector response measured at 210 nm. Chromatograms evaluated with

respect to retention time, resolution and peak shape.

Sibutramine eluted at 15 minutes and orlistat not eluted up to 25

minutes peak shape obtained was not proper, hence buffer changed.

2.5.5 Experiment No.5

The mobile phase was acetonitrile and a solution of 50 mM sodium

perchlorate buffer adjusted pH to 2.1 with 10% solution of perchloric

acid. Buffer was filtered through 0.45 μ filter and gradient program

applied as given in below table. The analytical column, a Zorbax SB

phenyl, 250 mm x 4.6 mm, 5 µ, maintained at temperature 25 °C. The

mobile phase flow rate was maintained at 1.5 ml/min. Standard

Sibutramine hydrochloride and orlistat solution was prepared at

48

concentration, 1200 μg/mL of orlistat and 100 μg/mL of sibutramine

hydrochloride in mobile phase. 20 μL standard solutions were injected

two times and average detector response measured at 210 nm.

Chromatograms evaluated according to retention time, resolution and

peak shape.

Table 2.02

Gradient Program for Experiment No.5

Sibutramine eluted in 11.4 minutes and orlistat not eluted up to 40

minutes, peak shape was proper and the forced degradation solutions

shown co-elution, hence tuning in gradient programme is required.

2.5.6 Experiment No.6

The mobile phase was acetonitrile and a solution of 50 mM sodium

perchlorate buffer adjusted pH to 2.1 with 10% solution of perchloric

Time (min) Buffer (%) Acetonitrile (%)

0 70 30

24 70 30

35 30 70

45 15 85

60 15 85

60 70 30

49

acid. Buffer was filtered through 0.45 μ membrane filter and applied

gradient program as given in below table. The analytical column, a

Zorbax SB phenyl, 250 mm x 4.6 mm, 5 µ, maintained at temperature 25

°C. The flow rate of mobile phase was maintained at 2.0 ml/min.

Standard Sibutramine hydrochloride and orlistat solution was prepared

at concentration, 1200 μg/mL of orlistat and 100 μg/mL of sibutramine

hydrochloride in mobile phase. 20 μL standard solutions were injected

two times and average detector response measured at 210 nm.

Chromatograms evaluated with respect to retention time, resolution and

peak shape.

Table 2.03

Gradient program for Experiment No.6

Time (min) Buffer (%) Acetonitrile (%)

0 60 40

20 40 60

25 20 80

35 20 80

40 60 40

45 60 40

Sibutramine hydrochloride and orlistat were eluted in 40 minutes

peak shape were proper and the forced degradation solutions shown a

well separation between the peaks.

50

The use of water instead of a solution of triethylamine and phosphoric

acid in the diluent mixture was verified and no modification in the

chromatographic profile or in the measured areas was observed.

2.5.7 Experiment No. 7

Applied Instrumental conditions given in experiment VI and injected

Orlistat known impurities. Impurities obtained at RT 3.793, 6.346,

24.417 and 33.287 were identified with known impurities spiking

method.

Impurities obtained at RT 6.644, 10.490 and 18.299 were isolated by

using preparative chromatographic technique and characterized these

isolated substance by using Mass spectroscopy, Neuclear magnetic

spectroscopy, IR, CHNS analyser and identified these impurities by

spiking method.

2.5.8 Experiment No.8 (Method Validation)

Specificity:

51

Diluent, standard solutions and sample solution of Sibutramine and

Orlistat capsules were prepared and injected into the HPLC as per

methology given in Experimental Results by using a photodiode detector.

A placebo solution of Sibutramine and Orlistat capsules was prepared

and injected into the HPLC along with selectivity solution as per

methology given Experimental results by using a photodiode array

detector.

The accelerated degradation conditions applied were: oxidant, acid

basic and light media. Sample were analysed against a freshly prepared

control sample. The peak purity was estimated using the tools of the

Waters software. Excipient solutions were used to the same degradation

conditions in order to demonstrate no interference. Specific details of the

experimental conditions for forced degradation are described below:

Effect of UV light:

1 ml of a solution of 1 mg/mL of sibutramine and 12 mg/mL of

orlistat in methanol was placed in a closed 1 cm quartz cell. The cells

were exposed to a UV chamber 100 x 18 x 17 cm with internal mirrors

and UV fluorescent lamp CRS F30W T8 emitting radiation at 254 nm for

15, 30, 60, 120 and 180 minutes. The same procedure was realized for

preparation for LC analysed samples, protected in aluminum foil (in

order to perotect from light) were submitted to identical conditions and

used as control. After the degradation treatment, the samples were

52

diluted to 100 μg/ml of sibutramine and 1200 μg/ml of orlistat with a

mixture of acetonitrile:methanol:water (6:3:1; v/v/v) and analysed

immediately.

Effect of Temperature (60 °C/24 h):

5 ml of a solution containing 1 mg/mL of sibutramine and 12 mg/mL

of orlistat in methanol was placed in a 10 ml volumetric flask at 60

°C/24 h for 24 h. After the degradation treatment, the samples were

diluted to 100 μg/ml of sibutramine and 1200 μg/ml of orlistat with a

mixture of acetonitrile:methanol:water (6:3:1; v/v/v) and analysed

immediately.

Effect of Humidity (25 °C/92% RH for 24 h):

5 ml of a solution containing 1 mg/mL of sibutramine and 12 mg/mL

of orlistat in methanol was placed in a 10 ml volumetric flask at 25

°C/92% RH for 24 h. After the degradation treatment, the samples were

diluted to 100 μg/ml of sibutramine and 1200 μg/ml of orlistat with a

mixture of acetonitrile: methanol: water (6:3:1; v/v/v) and analysed

immediately.

Effect of Oxidation:

Sibutramine and orlistat standards were dissolved in methanol (1

mg/ml of sibutramine amd12 mg/ml of orlistat) and 5 ml of this solution

53

was transferred to a volumetric flask, where hydrogen peroxide solution

(30%) was added until the final concentration of 10 %. The volume was

completed with methanol. After 20 hours the solution was diluted until

the final concentrations 100 μg/mL of sibutramine and 1200 μg/mL of

orlistst, filtered and analysed. Similar procedure was realized for the

capsules, when 25 ml of the initial solution 100 μg/ml of sibutramine

hydrochloride and 1200 μg/mL orlistat, obtained as described in sample

preparation for LC analysis, were transferred to a volumetric flask and

submitted to degradation. A control solution containing the excipients

was prepared under the same circumstances of the capsules.

Effect of Acid Hydrolysis:

5 ml of 1 mg/mL of sibutramine and 12 mg/mL of orlistat reference

standard solution was transferred to a volumetricflask and HCl was

added until the final concentration of 1M HCl. After 5 hours and 1 and 6

days, one aliquot of the solution was neutralized with NaOH 1M and

diluted with acetonitrile and water (40:60, v/v) until the final

concentration of 100 μg/ml of sibutramine and 1200 μg/ml of orlistat for

LC analysis. Similar procedure was realized with the capsules, when 25

ml of the initial solution 1 mg/mL of sibutramine and 12 mg/mL of

orlistat (obtained as described in sample preparation for LC analysis)

were transferred to a volumetric flask and submitted to the degradation

and diluted with acetonitrile and water (40:60, v/v) until the final

54

concentration of 100 μg/ml of sibutramine and 1200 μg/ml of orlistat for

LC analysis. A control solution containing the excipients was prepared

under the same circumstances of the capsules.

Effect of Alkaline Hydrolysis:

5 ml of 1 mg/mL of sibutramine and 12 mg/mL of orlistat reference

standard solution was transferred to a volumetric flask and NaOH

(alkaline degradation) was added until the final concentration of 1M

NaOH. After 5 hours and 1 and 6 days, one aliquot of the solution was

neutralized with HCl 1M and diluted with acetonitrile and water (40:60,

v/v) until the final concentration of 100 μg/ml of sibutramine and 1200

μg/ml of orlistat for LC analysis. Similar procedure was realized with the

capsule, when 25 ml of the initial solution 1 mg/mL of sibutramine and

12 mg/mL of orlistat (obtained as described in sample preparation for LC

analysis) were transferred to a volumetric flask and submitted to the

degradation and diluted with acetonitrile and water (40:60, v/v) until the

final concentration of 100 μg/ml of sibutramine and 1200 μg/ml of

orlistat for LC analysis. A control solution containing the excipients was

prepared under the same circumstances of the capsules.

LOD and LOQ:

55

The qualification and detection limits were obtained based on signal-

to-noise approach. The background noise was obtained after injection of

the blank, observed over a distance equal to 20 times the width at half-

height of the peak in a chromatogram obtained by the injection 2 μg/mL

of each reference standards. The signal-to-noise ratio applied was 10:1

for the LOQ and 3:1 for LOD. The results were verified experimentally.

Based on the determination of signal to noise ratio and visual

observation for known impurities, six replicate injections were made for

LOD &LOQ.

Linearity and Range:

To test linearity, standard plots were construted with six

concentrations in the range 0.13-15.35 μg/mL of 4-chlorophenyl

acetonitrile, 0.12-14.84 μg/mL of Amine impurity, 0.11-14.67 μg/mL of

Nitrile impurity, 0.11-14.51 μg/mL of Ketone impurity, 0.81-88.80

μg/mL of ODI, 2.56-91.80 μg/mL of NNHUI, 0.95-91.80 μg/mL of ODLI,

5.07-90.84 μg/mL of CDZI in triplicates. The linearity was evaluated by

linear regression analysis that was calculated by the least square

regression.

Accuracy:

56

The accuracy was determined by the recovery of known amounts of 4-

chlorophenyl acetonitrile, Amine impurity, Nitrile impurity, and Ketone

impurity, ODI, NNJUI, ODLI and CDZI to the Sample in the beginning of

the preparative process. The added levels were LOQ, 50, 100 and 150%

of the specified limit in triplicate and then proceed with sample

preparation as described under experimental result.

Precision:

Six replicate injections of the standard preparation were madeinto the

HPLC used the methodology given in experimental result.

Six spiked sample preparations and one control sample preparation of

Sibutramine and Orlistat capsules were prepared and injected into the

HPLC using the method as described under experimental result.

Ruggedness:

Six spiked sample preparations and one control sample preparations

of Sibutramine and Orlistat capsules were analysed by a different

analyst, using different column, on different day and injected into a

different HPLC using the method as described in experimental result,

along with standard preparation.

Robustness:

57

Standard preparation, diluent, placebo preparation and sample

preparation in triplicate of the sample of Sibutramine and Orlistat

capsules were prepared as described in experimental result. The samples

along with standard and placebo were injected under different

chromatographic conditions.

Stability of analytical solution:

Standard solution, Sample solution were analysed initially and at

different time intervals at room temperature.

Systemsuitability:

The system suitability was verified through the evaluation of the

obtained parameters for the standard elution, such as theoretical plates,

peak asymmetry and retention factor, verified in different days of the

method validation.

2.6 EXPERIMENTAL RESULTS

On the basis of Sibutramine hydrochloride and Orlistat Analytical

method development experimental trials, RP-HPLC method described in

experiment No.7 was suitable for simultaneous determination of

Sibutramine hydrochloride and orlistat impurities in a single method.

RS method chromatographic conditions were applied for Identification as

well as quantification of related substance. Impurities were identified by

spiking known impurities in to sample preparation. Preparation of stock

58

solutions: Prepare solution having the concentration of sibutramine

hydrochloride 100 ppm and Orlistat 1200 ppm, in acetonitrile. Mix

standard solution: Prepared a solution having the concentrations of

sibutramine hydrochloride 100 ppm and orlistat 1200 ppm.

Sample Preparation:

Twenty capsules were weighed and crushed finely, powder equivalent

to 240 mg of Orlistat and 20 mg sibutramine hydrochloride (480mg) was

transferred to 200 ml volumetric flask and added 150 ml of diluent,

shake for 10 min and made up to volume with diluent, filtered the above

sample through 0.45 µ Teflon filter and injected. Concentration of

sibutramine hydrochloride was 100 ppm and orlistat was 1200 ppm.

Separately injected equal volumes of diluent, standard preparation in

six replicates and sample twice in to equilibrated HPLC system and

record chromatograms and measured the response in terms of peak area.

System suitability parameters occurred during method validation were

Theoretical plates not less than 8000, tailing factor not more than 1.5,

relative standard deviation for six replicates of standard solution is not

more than 2.0%.

2.7 DISCUSSION OF RESULTS:

LOD and LOQ:

59

RSD is less than 33% at LOD level and less than 10% at LOQ level

for sibutramine, orlistat and known impurities.

Linearity and range:

The correlation coefficients are less than 0.9995 for Sibutramine,

Orlistat and known Impurities.

Precision:

system precision RSD is less than 5% and method precision RSD is

less than 10% for sibutramine, orlistat and known impurities.

Accuracy:

The mean recoveries for sibutramine, orlistat and known impurities

are within 90 -110 %.

Specificity:

Retention time of Sibutramine and Orlistat peaks and known peaks

in sample preparation is comparable with respect to retention time of

Sibutramine and orlistat and known impurities peaks in standard

preparation. Peak purity passes for Sibutramine and orlistat and known

impurities peaksin standard and sample preparations. No intereference

was observed at the retention time of Sibutramine and orlistat and

known impurities peaks. Peak purity passes for all degradation

conditions.

60

Ruggesness:

The RSD of twelve results obtained from two different analysts are

within 10 %.

Robustness:

Sibutramine, orlistat and all known impurities peaks were resolved

with each other and system suitability complies for all variable

conditions, the test method is robust for all variable conditions.

Stability in Analytical solution:

Standard and sample solutions are stable for 12 h at room

temperature

System Suitability:

Theoretical plates are less than 2000, tailing factor is less than 2.0

and relative standard deviation is less than 5.0 for six standard replicate

injections.

Table 2.04

Peak purity data for Orlistat and Sibutramine HCl

Sr. No. Name

Purity

Criteria

61

1 Sibutramine hydrochloride in

standard solution Pass

2 Sibutramine hydrochloride in

sample solution Pass

3 Orlistat in standard solution Pass

4 Orlistat in sample solution Pass

Table 2.05

RT, RRT, LOD and LOQ of Sibutramine Impurities

Name of Compound RT

(min) RRT RRF LOD

(μg/mL) LOQ

(μg/mL)

Sibutramine HCl 9.93 1.00 1.00 0.038 0.115

4 CPA 6.67 0.67 0.95 0.040 0.121

Amine impurity 7.92 0.80 1.00 0.038 0.115

Nitrile impurity 11.28 1.14 1.02 0.037 0.113

Ketone impurity 19.82 2.00 1.10 0.035 0.105

Table 2.06

RT, RRT, LOD and LOQ of Orlistat Impurities

Name of Compound RT

(min) RRT RRF LOD

(μg/mL) LOQ

(μg/mL)

62

Orlistat 30.82 1.00 1.00 0.32 0.99

Dimeric impurity 5.39 0.17 1.23 0.26 0.80

NN Dicyclohexyl urea 6.76 0.22 0.40 0.80 2.48

Delactone impurity 25.47 0.83 1.08 0.30 0.92

CBZ impurity 35.22 1.14 0.20 1.60 5.07

Table 2.07

Linearity Data of Orlistat and Sibutramine HCl at Limiting Conc.

Level (%) Orlistat Sibutramine HCl

63

Conc.

(µg/mL)

Area Conc.

(µg/mL)

Area.

LOQ 1.01 2023 0.12 2043

50 30.04 58998 5.33 86548

80 48.06 96501 8.52 140284

90 54.06 106842 9.59 155317

100 60.07 119550 10.65 173791

110 66.08 131768 11.72 191552

120 72.08 142312 12.78 206881

150 90.11 180257 15.98 262042

Slope 1993 16335

Intercept -211 -144

Correlation

coefficient

0.9999 0.9999

Table 2.08

Linearity of Sibutramine HCl Impurities

Level 4 CPA SAI SNI SKI

64

Conc.

(µg/mL) Area

Conc.

(µg/mL) Area

Conc.

(µg/mL) Area

Conc.

(µg/ml) Area

LOQ 0.13 2169 0.12 2125 0.11 1894 0.11 2112

50 5.12 81725 4.95 86014 4.89 85807 4.84 94725

80 8.18 132609 7.91 139424 7.82 139232 7.74 153548

90 9.21 147403 8.90 154046 8.80 153204 8.70 170677

100 10.23 165101 9.89 173241 9.78 173347 9.67 191170

110 11.25 183791 10.88 192661 10.76 192970 10.64 212600

120 12.28 197527 11.87 205602 11.74 205312 11.60 227798

150 15.35 251416 14.84 262980 14.67 263973 14.51 288476

Slope 16288 17607 17837 19836

Intercept -756 -547 -812 -413

C.C 0.9998 0.9998 0.9997 0.9999

65

Table 2.09

Linearity of Orlistat Impurities

Level ODI (%) NNHUI (%) ODLI (%) CBZI (%)

Conc.

(µg/mL) Area

Conc.

(µg/mL) Area

Conc.

(µg/mL) Area

Conc.

(µg/ml) Area

LOQ 0.81 2021 2.56 2004 0.95 2024 5.07 2017

50 29.60 72788 30.60 23671 30.60 64105 30.28 11847

80 47.36 118108 48.96 38409 48.96 103911 48.45 19205

90 53.28 131283 55.08 42263 55.08 114808 54.50 21347

100 59.20 147047 61.20 47820 61.20 129114 60.56 23910

110 65.12 163692 67.32 53233 67.32 143588 66.62 26590

120 71.04 175926 73.44 56638 73.44 153232 72.67 28491

150 88.80 223922 91.80 72820 91.80 195995 90.84 36080

Slope 2507 786 2121 396

Intercept -674 -229 -406 -49

C.C 0.9998 0.9997 0.9998 0.9999

66

Table 2.10 Recovery of Orlistat impurities at LOQ Level

ODI (%) NNHUI (%) ODLI (%) CBZI (%)

LOQ Spl. 1 103.9 98.3 105.5 95.5

LOQ Spl. 2 98.7 96.8 100.2 98.8

LOQ Spl. 3 101.6 103.5 101.9 102.4

Mean 101.4 99.5 102.5 98.9

S.D 2.6 3.5 2.7 3.5

%RSD 2.6 3.5 2.6 3.5

Table 2.11

Recovery of Orlistat Impurities at 50, 100 and 150 % Level

ODI (%) NNHUI (%) ODLI (%) CBZI (%)

50 Spl. 1 102.3 103.4 101.4 100

50 Spl. 2 99.5 102.7 101.1 100.8

50 Spl. 3 101.8 104.3 101.6 99

100 Spl. 1 98.3 99.4 98.6 100.4

100 Spl. 2 98 99.8 98.6 99.6

100 Spl. 3 98.9 98.3 96.9 99.2

150 Spl. 1 98.5 97.2 99.6 98.7

150 Spl. 2 101.2 99.2 97.3 99.7

150 Spl. 3 98.1 98.3 98.3 98.9

Mean 99.62 100.29 99.39 99.59

S.D 1.69 2.53 1.84 0.71

%RSD 1.70 2.52 1.85 0.72

67

Table 2.12 Recovery of Sibutramine HCl Impurities at LOQ Level

4-CPA (%) SAI (%) SNI (%) SKI (%)

LOQ Spl. 1 101.3 101.3 103.5 103.4

LOQ Spl. 2 99.7 99.8 102.2 99.5

LOQ Spl. 3 101.6 102.5 100.9 100.6

Mean 100.9 101.2 102.2 101.2

S.D 1.0 1.4 1.3 2.0

%RSD 1.0 1.3 1.3 2.0

Table 2.13 Recovery of Sibutramine HCl Impurities at 50,100 and 150%

Level

4-CPA (%) SAI (%) SNI (%) SKI (%)

50 Spl. 1 101.3 102.4 103.4 99.5

50 Spl. 2 99.5 104.7 100.1 101.46

50 Spl. 3 102.8 104.3 100.6 101.5

100 Spl. 1 99.3 95.4 99.6 100.4

100 Spl. 2 99 96.8 97.6 99.5

100 Spl. 3 98.9 99.3 99.9 98.8

150 Spl. 1 102.4 99.2 99.6 99.2

150 Spl. 2 101.2 99.7 97.3 101.3

150 Spl. 3 99.1 99.3 97.3 102.3

Mean 100.39 100.12 99.76 100.44

S.D 1.55 3.15 1.88 1.24

%RSD 1.54 3.14 1.89 1.24

68

Table 2.14

System Precision Data for Sibutramine Impurities

SH

(Area)

4-CPAI

(Area)

SAI

(Area)

SNI

(Area)

SKI

(Area)

Rep.1 173791 165101 173241 173347 191170

Rep. 2 172341 165098 174211 172234 191234

Rep. 3 173542 164981 174842 174121 188187

Rep. 4 173987 164892 173451 175236 188456

Rep. 5 174721 166234 173562 174675 189241

Rep. 6 172981 165990 173245 173425 189234

Mean (Area) 173561 165383 173759 173840 189587

S.D 825.24 575.47 639.28 1069.53 1319.24

% RSD 0.48 0.35 0.37 0.62 0.70

Table 2.15

System Precision Data for Orlistat Impurities

Orlistat (Area)

ODI (Area)

NNDHU (Area)

ODLI (Area)

CBZI (Area)

Rep.1 119550 47820 147047 129114 23910

Rep. 2 116341 47342 147745 128234 23923

Rep. 3 117825 47394 147253 129841 23876

Rep. 4 119145 47209 147846 129244 23985

Rep. 5 117871 47645 147980 128643 23908

Rep. 6 115629 47652 147823 128543 23890

Mean(Area) 117727 47510 147616 128937 23915

S.D 1528.86 230.98 374.28 579.43 37.90

%RSD 1.30 0.49 0.25 0.45 0.16

69

Table 2.16 Method Precision Data for sibutramine HCl impurities

SH (%) 4 CPA (%) SAI (%) SNI (%)

Spl. 1 1.01 1.06 1.15 1.15

Spl. 2 1.03 1.04 1.19 1.11

Spl. 3 1.01 1.02 1.14 1.12

Spl. 4 0.991 1.13 1.17 1.09

Spl. 5 0.987 1.19 1.15 1.08

Spl. 6 0.989 1.09 1.17 1.1

Mean (%) 1.003 1.088 1.162 1.108

S.D 0.017 0.063 0.018 0.025

%RSD 1.68 5.79 1.58 2.24

Table 2.17

Intermediate Precision Data for Sibutramine HCl Impurities

SH (%) 4 CPA (%) SAI (%) SNI (%)

Rep.1 1.05 1.09 1.05 1.11

Rep. 2 1.03 1.14 1.09 1.01

Rep. 3 1.05 1.12 1.17 1.02

Rep. 4 1.01 1.14 1.13 1.09

Rep. 5 1.07 1.19 1.14 1.08

Rep. 6 0.989 1.09 1.16 1.08

Mean (%) 1.033 1.128 1.123 1.065

S.D 0.030 0.038 0.045 0.040

%RSD 2.88 3.34 4.05 3.79

Table 2.18

70

Method Precision Data for Orlistat Impurities

ODI (%) NNDHU (%) ODLI (%) CBZI (%)

Spl. 1 1.04 1.05 1.12 1.13

Spl. 2 1.02 1.08 1.11 1.12

Spl. 3 1.03 1.07 1.1 1.14

Spl. 4 1.01 1.11 1.13 1.07

Spl. 5 1.07 1.12 1.16 1.18

Spl. 6 1.09 1.11 1.11 1.11

Mean (%) 1.043 1.090 1.122 1.125

S.D 0.031 0.028 0.021 0.036

%RSD 2.95 2.53 1.91 3.22

Table 2.19 Intermediate precision Data for Orlistat Impurities

ODI (%) NNDHU (%) ODLI (%) CBZI (%)

Spl. 1 1.11 1.03 1.12 1.13

Spl. 2 1.12 1.09 1.09 1.05

Spl. 3 1.05 1.1 1.13 1.04

Spl. 4 1.08 1.08 1.13 1.09

Spl. 5 1.09 1.09 1.09 1.08

Spl. 6 1.11 1.11 1.13 1.09

Mean (%) 1.093 1.083 1.115 1.080

S.D 0.026 0.028 0.020 0.032

%RSD 2.36 2.59 1.77 2.99

71

Table 2.20 Solution Stability Data of Orlistat impurities

ODI (%) NNDHU (%) ODLI (%) CBZI (%)

Initial 1.03 1.03 1.01 1.05

After 8 h 1.05 1.04 1.05 1.08

After 12 h 1.08 1.09 1.05 1.09

After 24 h 1.03 1.02 1.03 1.03

After 48 h 1.18 1.07 1.12 1.08

Mean (%) 1.074 1.05 1.052 1.066

S.D 0.063 0.029 0.041 0.025

%RSD 5.84 2.78 3.94 2.35

Table 2.21

Solution stability Data of Sibutramine HCl Impurities

SDI (%) NNDHU (%) SDLI (%) CBZI (%)

Initial 0.98 1.08 1.06 1.08

After 8 h 1.01 1.1 1.07 1.02

After 12 h 1.03 1.09 1.02 1.09

After 24 h 1.08 1.06 1.07 1.02

After 48 h 1.1 1.02 1.13 1.08

Mean (%) 1.04 1.07 1.07 1.058

S.D 0.049 0.032 0.039 0.035

%RSD 4.76 2.96 3.68 3.30

72

Figure 2.03

Amine Impurity HMBC NMR Spectrum-1

73

Figure 2.04

Amine Impurity HMBC NMR Spectrum-2

74

Figure 2.05

Amine Impurity HMBC Nitrogen NMR Spectrum-3

75

Figure 2.06

4- Chloro phenyl acetonitrile HMBC NMR Spectrum-1

76

Figure 2.07

4- Chloro phenyl acetonitrile HMBC NMR Spectrum-2

77

Figure 2.08

Nitrile Impurity HMBC NMR Spectrum-1

78

Figure 2.09

Nitrile Impurity HMBC NMR Spectrum-2

79

Figure 2.10

4- Chloro Acetonitrile Impurity C-13 NMR Spectrum-1

80

Figure 2.11

Amine Impurity C-13 NMR Spectrum-1

81

Figure 2.12

4-Chloro acetonitrile C-13 NMR Spectrum-2

82

Figure 2.13

Ketone Impurity C-13 NMR Spectrum-1

Figure 2.14

83

Ketone Impurity C-13 NMR Spectrum-2

84

Figure 2.15

Nitrile Impurity C-13 NMR Spectrum-1

85

Figure 2.16

Nitrile Impurity C-13 NMR Spectrum-2

86

Fig. 2.17

4- Chloro phenyl acetonitrile Proton NMR Spectrum-1

87

Figure 2.18

Amine Impurity proton NMR spectrum-1

Figure 2.19

88

Amine Impurity Proton NMR Spectrum-2

Figure 2.20

89

Ketone Impurity Proton NMR Spectrum-1

Figure 2.21

90

Ketone Impurity Proton NMR Spectrum-2

Figure 2.22

91

Nitrile Impurity Proton MNR Spectrum-1

92

Figure 2.23

Nitrile Impurity Proton MNR Spectrum-2

93

Figure 2.24

Amine Impurity Mass Spectrum-1

94

Figure 2.25

4-ChloroPhenyl Acetonitrile Mass Spectrum-1

Figure 2.26

95

Amine Impurity Mass Spectrum-1

Figure 2.27

96

Ketone Impurity Mass Spectrum-1

97

Figure 2.28

Nitrile Impurity Mass Spectrum-1

98

Figure 2.29

Sibutramine + Orlistat Capsules Placebo Chromatograph

Figure 2.30

Sibutramine + Orlistat Capsules Sample Chromatograph

99

Figure 2.31

Mix Impurities Standard Chromatograph

Figure 2.32

Mix Impurities Standard Chromatograph

100

Figure 2.33

Impurities Spiked Sample Chromatograph

Figure 2.34

101

Ketone Impurity UV Spectrum

Figure 2.35

102

Nitrile Impurity UV Spectrum

Figure 2.36

103

4-Chlorophenyl Acetonitrile UV Spectrum

2.8 Summary, Conclusion and Recommendations.

104

The reversed phase LC method proposed was found to be simple, fast,

accurate, precise, linear, robust and specific and it is powerful tool to

investigate chemical stability of sibutramine and orlistat. The robustness

of the method was verified with small variation on pH, concentration of

triethylamine solution, concentration of organic phase, detector

wavelength, column manufacturer and analysis temperature. All the

parameters meet the criteria of the ICH guidelines for method validation

and its chromatographic retention time of 60 min allows the analysis of a

large number of samples in an adequate period of time. The method

could therefore be recommended for routine quality control analysis of

raw material and of capsules, as well as for routine quality control

analysis of raw material and of commercial cream, as well as for

protocols of sibutramine and orlistat stability study.