International Bulletin of Drug Research.

, 1(2): 71-80

METHOD DEVELOPMENT AND VALIDATION OF

RIFAMPICINE AND PIPERINE IN THEIR
COMBINED DOSAGE FORM
KAUSHIK G. KAPURIYA*1, PRAKASH M. PARMAR1, HIRAL R. TOPIYA1,
SHITAL D. FALDU1
________________________________________________________________________
ABSTRACT
The RP-HPLC method has been developed for simultaneous estimation of Rimfapicin And
Piperine in Their Combined Dosage Form. For RP-HPLC method all the standard and
sample solutions were prepared in Methanol: Acetonitrile. A stability indicating reversedphase HPLC method has been developed and subsequently validated for simultaneous
estimation of Rimfapicin and Piperine in their combination product. The proposed RP-HPLC
method utilizes a Hypersil BDS C18 (25cm x 4.6mm, 5 m) column, mobile phase consisting
of Buffer and Acetonitrile in the proportion of 55 : 45 (v/v) with apparent pH adjusted to
6.8, and UV detection at 341 nm.. The described method was linear over a range of 824 g/ml for Rifampicin and 0.4-1.2 g/ml for Piperine. The mean recoveries were 99.91 and
100.53% for Piperine and Rifampicine, respectively. Validations of the proposed method
were carried out for its accuracy, precision, linearity and range, specificity, LOD and LOQ
according to ICH guidelines. The method was validated by evaluation of different
parameters. The method has been successfully applied for the analysis of drugs in
pharmaceutical formulation.
KEYWORDS
Reverse Phase High Performance Liquid Chromatography, Rifampicine and Piperine.
AFFILIATION
1.

*Mr. Kaushik G. Kapuriya, Assistant Professor, Smt. R. D. Gardi B. Pharmacy

College. Nyara, Dist: Rajkot, 360110, Gujarat, India.

2.

Prakash M. Parmar, Research Scholar, RDGPC, Nyara, Rajkot, Gujarat, India.

3.

Ms. Hiral R. Topiya, Assistant Professor, RDGPC, Nyara, Rajkot, Gujarat, India.

4.

Dr. Sheetal Faldu, Principal, RDGPC, Nyara, Rajkot, Gujarat, India.

5.

*Author for correspondence: Mob no: 7600027116, email: kkapuriya@gmail.com

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International Bulletin of Drug Research., 1(2): 71-80

INTRODUCTION
Rifampicin (RIFA) is a well known Anti-Tuberculosis drug.1 Methods for Estimation of
Rifampicin is official in IP2, BP3 and USP4. Literature survey reveals HPLC5, HPTLC6 and
Visible Spectrophotometry methods7 for determination of RIFA in single and RP-HPLC,8, 9
Visible Spectrophotometry10,11 and HPTLC12 methods in combined pharmaceutical dosage
forms as well as in biological fluids.
Piperine (PIPE) is a natural alkaloid which is used as Bio enhancer13. Piperine is official in
IP14. Literature survey reveals RP-HPLC15, UV Spectrophotometry and HPTLC method for
the determination of Piperine in single and in combination with other drugs. The combined
dosage forms of RIFA and PIPE along with Isoniazid are available in the market and used as
anti tuberculosis drugs.
The simultaneous estimation of Rimfapicin and Piperine in their combined dosage form is not
official in any pharmacopoeia; hence no official method is available for the estimation of
RIFA and PIPE in their combined dosage forms. Literature survey reveals simple
spectrophotometric method for simultaneous estimation of RIFA and PIPE in combined
dosage forms. Literature survey may not reveal any RP-HPLC for the estimation of RIFA and
PIPE in their combined dosage form.
The proposed RP HPLC method was found to be sensitive, accurate and precise for
determination of RIFA and PIPE in capsule dosage form. The method utilizes easily available
and cheap solvent for analysis of RIFA and PIPE hence the method was also economic for
estimation of RIFA and PIPE from capsule dosage form. Hence it can be conveniently
adopted for routine quality control analysis of the drugs in combined pharmaceutical
formulation.
EXPERIMENTAL SECTION
Apparatus and instruments
The HPLC system (Shimadzu VP, Japan), consisted of a system controller (SCL-10AVP), online degasser (DGU-14A), low-pressure gradient flow control valve (FCV-10ALVP), solvent
delivery module (LC-10ADVP), auto injector (SIL-10 ADVP), column oven (CTO-10AVP), UV
VIS and PDA detector (SPD-10AVP) and CLASS VP software version 6.14 SP1. Sonicator
(5510, Branson Ultrasonics Corporation, USA) was used in the study.
Material
RIFA and PIPE bulk powder was gifted by Cadila Pharmaceuticals Ltd. Dholka, Ahmadabad,
and Gujarat, India. The commercial fixed dose combination product was procured from the
local market. All other reagents used were of HPLC grade.
Method Development and Optimization
Selection of detection wavelength
The standard solution of Rifampicin and Piperine were scanned over the range of 200 nm to
400 nm wavelengths. The overlay spectra were recorded and the intersection point

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International Bulletin of Drug Research., 1(2): 71-80

wavelength was selected. It was observed at 341 nm. So the wavelength, 341 nm, was
selected for the determination of Rifampicin and Piperine.
Selection of mobile phase
The mobile phase was selected on the basis of best separation, peak purity index, peak
symmetry, theoretical plate etc. So no. of trials was taken for the selection of mobile phase.
Generally As per I.P2007 method for Rifampicin and various combination of Rifampicin, a
combination of Buffer (1.4 gm Disodium Hydrogen Orthophosphate in 1000 ml water, pH
6.8 adjusted with dilute Phosphoric acid) with Acetonitrile was used as Mobile phase. So also
for this combination of Rifampicin and Piperine, the same mobile phase in different ratio was
used to optimize best result. Also different solvent mixtures were used to optimize best result.
Selection of pH
In mildly basic aqueous solutions (pH 8.2, 20-22C) in the presence of air, Rifampicin is
converted to rifampin quinine. Under basic conditions Rifampicin undergoes desacetylation
at 22C forming the 25-desacetylrifampin. Rifampicin decomposes rapidly in acidic or
alkaline conditions at 25C but slowly in neutral conditions so it is best to prepare aqueous
solutions with oxygen-free solvent and at neutral pH. So pH 6.8 was adjusted with dilute
Phosphoric acid.
Selection of column
For HPLC, various columns are available, but C18 column was preferred over other columns.
Hypersil BDS C18 column (250 mm x 4.6 mm i.d., 5 m particle size) was chosen to give
good peak shape, good lifetime and high resolution compared to other C18 columns.
Selection of column temperature
The column temperature (30C) has minimized variation of retention and made the peak
sharp. It has also shortened the run time.
Chromatographic Conditions
-

Column Hypersil BDS C18 (25cm x 4.6mm, 5 m)

Column temp. - 25C

Sample temp. - 10C

Buffer- 1.4 gm Disodium Hydrogen Orthophosphate in 1000 nl water, pH 6.8

adjusted with dilute Phosphoric acid

Mobile Phase - Buffer : Acetonitrile (55 : 45)

Solvent Mixture - Methanol : Acetonitrile ( 50 : 50 )

Standard Solution - (A) Piperine 50 mg dissolve and dilute upto 100 ml with
solvent mixture

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International Bulletin of Drug Research., 1(2): 71-80

-

(B) 20 mg Rifampicin Dissolve in 70 ml Solvent mixture 2 ml solution A

Dilute up to 200 ml with solvent mixture (Rifampicin 100 g/ml & Piperin 5 g/ml)

Flow Rate 1.5 ml per minute

Detector Condition 341 nm

Injection Volume 20 l

Buffer Preparation
Dissolve 1.42 g of disodium hydrogen phosphate anhydrous in 1000 ml of water. Adjust pH
to 6.8 with orthophosphoric acid.
Diluent
Prepare filtered and degassed mixture of methanol and acetonitrile in ratio (50:50).
Piperine standard stock preparation:
Accurately weigh and transfer 50.0 mg of Piperine working standard of known potency into
100 ml volumetric flask. Add 70 ml of diluent and sonicate to dissolve. Dilute up to mark
with diluents and mix.
Standard Solution preparation
Accurately weigh and transfer 20.0 mg of Rifampicin working standard of known potency
into 200 ml amber colored volumetric flask and add 70 ml of diluent. Sonicate to dissolve,
add 2.0 ml of Piperine standard stock solution and make up to mark with diluent and mix.

Figure 1. Chromatograph of standard RIFA and PIPE

Table 1. Data for system suitability test for Rifampicin and Piperine
Parameters

Rifampicin RSD
(n = 6)

Piperine RSD
(n = 6)

Retention time (min)

2.75 0.1%

8.11 0.2%

Tailing factor

1.1 0.3%

1.0 0.6%

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International Bulletin of Drug Research., 1(2): 71-80

Theoretical plates

5631 0.7%

13218 0.8%

Area

1922490 0.5%

480780 0.2%

Resolution

8.93 0.8%

Assay Preparation
Weigh 20 capsules and determine average net content of blend. Reserve Isoniazid tablet for
assay of Isoniazid. Accurately weigh and transfer quantity of capsule contents equivalent to
about 200.0 mg of Rifampcin into 200 ml amber colored volumetric flask. Add 100 ml of
diluent and sonicate for about 20 minutes. Dilute to volume with diluent and mix. Filter
resulting solution with 0.45 m PVDF filter discarding first few ml of filtrate. Further dilute
5.0 ml of this solution to 50.0 ml with diluents and mix.
Chromatographic Parameters
Liquid chromatograph is equipped with PDA detector or variable wavelength UV detector, an
injector and data processor.
-

Column Hypersil BDS C18 (25cm x 4.6mm, 5 m)

Column temperature - 25C

Sample temperature - 10C

Flow Rate 1.5 ml per minute

Detector Condition 341 nm

Injection Volume 20 l

Retention time Rifampicin: About 3.5 minutes.

Piperine: About 7.0 minutes.
RESULTS AND DISCUSSION

Selection of Wavelength
The detection was carried out in the UV region and wavelength selected for detection was
341 nm in mobile Phase.
Validation of RP-HPLC Method
As per the ICH guidelines16, the method validation parameters checked were linearity,
accuracy, precision, limit of detection, limit of quantitation.
Linearity and Range
The linearity of the method was determined for the formulation at five concentration levels
ranging from 8 to 24 g/ml for RIFA and 0.4- 1.2 g/ml PIPE. The equation for regression

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line was y = 23308X-1898.2 (R2= 1) for RIFA and y = 105066X-372.4 (R2= 0.999) for
PIPE.
Linearity of Rifampicin
y = 23308x - 1898.2
R2 = 1

600000

Average area

500000
400000
300000

area

200000

Linear (area)

100000
0
0

10

20

30

Concentration

Figure 4. Calibration curve for Rifampicin

Linearity of Piperine
y = 105066x - 372.4
R2 = 0.9999

140000

Average area

120000
100000
80000

area

60000

Linear (area)

40000
20000
0
0

0.5

1.5

Concentration

Figure 5. Calibration curve for Piperine

Table 2. Linearity of Rifampicin and Piperine by RP-HPLC method
Concentration (g/ml)

Mean area (n=3)

Linearity level
Rifampicin

Piperine

Rifampicin

Piperine

Level-1 (50%)

0.4

184338

41536

Level-2 (75%)

12

0.6

278440

62432

Level-3 (100%)

16

0.8

370801

84311

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International Bulletin of Drug Research., 1(2): 71-80

Level-4 (125%)

20

1.0

463716

104607

Level-5 (150%)

24

1.2

557862

125514

Correlation coefficient

1.0000

0.9999

Slope of regression line

23308.1

105065.5

Y-intercept

-1898.2

-372.4

The results show that an excellent correlation exists between response factor and
concentration of drugs within the concentration range indicated above.
Accuracy and Precision
The accuracy of the method was determined by recovery experiments. The recovery studies
were carried out at three levels of 50, 100 and 150% and the percentage recovery was
calculated and presented in Table 3.
Table 3. Recovery studies of Rifampicine (RIFA) and Piperine (PIPE)
% Mean Recovery

Level of %
Recovery

% RSD

RIFA

PIPE

RIFA

PIPE

50

100.1

99.97

0.14

0.12

100

100.2

100.1

0.18

0.23

150

100.1

100.2

0.16

0.24

Table 4. Intraday precision data for analysis of Rifampicin and Piperine

Drug

Rifampicin

Piperine

Concentration (g/ml)

Mean area RSD

50

1153497 0.054

100

2320522 0.049

150

3491052 0.051

2.5

260639 0.071

529052 0.056

7.5

788648 0.062

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Table 5. Interday precision data for analysis of Rifampicin and Piperine
Drug

Rifampicin

Piperine

Concentration
(g/ml)

Mean area RSD

50

1152086 0.153

100

2318678 0.078

150

3490432 0.172

2.5

260608 0.108

528969 0.080

7.5

788593 0.184

RSD value ranges for Rifampicin it is 0.049 0.054% for intraday, 0.078 0.172% for
interday and for Rifampicin is 0.056 0.071% for intraday, 0.080 0.184% for interday. The
results obtained accurately fall within the limit of acceptance criteria. Hence, the method can
be termed as precise.
Limit of detection and limit of qualification
The limit of detection (LOD) and limit of quantification (LOQ) were calculated using signalto-noise (i.e. 3.3 for LOD and 10 for LOQ) ratio using following equations designated:
LOD = 3.3 /S
LOQ = 10 /S
Where, = the standard deviation of the response,
S = slope of the calibration curve.
LOD and LOQ for Rifampicin are found to be 0.498 g/ml and 1.51g/ml respectively. LOD
and LOQ for Piperine are found to be 0.081 g/ml and 0.246 g/ml.
CONCLUSION
From the results obtained, it is obvious that the proposed method is applicable for the
determination of Rifampicin and Piperine without interference and with good sensitivity. The
results obtained indicate that the proposed method for the estimation of Rifampicin and Piperine
is simple, specific, rapid, linear, accurate, precise, robust, sensitive and suitable for intended use.
These merits suggest the use of the proposed method in routine and quality control analysis of
Rifampicin and Piperine without interference from commonly encountered excipients and
additives.

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