Journal of Applied Pharmaceutical Science Vol. 8(03), pp.

072-081, March, 2018

Available online at http://www.japsonline.com
DOI: 10.7324/JAPS.2018.8311
ISSN 2231-3354

Effect of Piperine on Pharmacokinetics of Rifampicin and

Isoniazid: Development and Validation of High Performance
Liquid Chromatography Method

Amit Singh , Smita Verma , Nouh M H Al Jarari , Ajay Pal Singh , Neeraj Kumar Fuloria , Shivkanya Fuloria ,
1* 2 3 4 5 5

Pradeep Kumar Sharma , Chhotelal

6 6

1
School of Pharmacy, Monad University, Hapur, Uttar Pradesh, 245101, India.
2
Department of Chemistry, National Institute of Pharmaceutical Education and Research (NIPER), Kolkata, West Bengal, India.
3
Department of Pharmacology, Faculty of Medicine, University of Benghazi, Libya.
4
HIMT College of Pharmacy, Knowledge Park, Phase-II, Greater Noida, Gautam Budh Nagar, Uttar Pradesh, India.
5
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, AIMST University, Semeling Campus, Jalan Bedong-Semeling, Bedong, Kedah Darul
Aman, 08100, Malaysia.
6
R. V. Northland Institute, Dadri, Gautam Budh Nagar, Uttar Pradesh, 203 207, India.

ARTICLE INFO ABSTRACT

Article history: In the present investigation a method for simultaneous determination of rifampicin, isoniazid and piperine by liquid
Received on: 23/05/2017 chromatography has been developed and optimized in terms of specificity, accuracy, precision, sensitivity and
Accepted on: 21/12/2017 stability. The purpose of this investigation concerns the effect of piperine on the pharmacokinetic of rifampicin and
Available online: 30/03/2018 isoniazid in rat model. Suspension of rifampicin, isoniazid and piperine were administered orally in Swiss albino
rat and the drug concentration in plasma was estimated by high performance liquid chromatography. The chromatic
separation of rifampicin, isoniazid and piperine were achieved by carrying out on a Zorbax Eclipse XDP C18 column
Key words:
(150 mm × 4.6 mm × 5 µm) using potassium dihydrogen phosphate (pH 4.5) and acetonitrile as the mobile phase
Pharmacokinetic, Rifam-
(30:70) at a flow rate of 0.8 mL/min with detection at 270 nm. After oral administration the observed pharmacokinetic
picin, Isoniazid, Piperine,
parameter of rifampicin along with piperine indicates significant enhancement in Cmax and area under the curve (AUC),
Bioavailability, Reverse
while in isoniazid with piperine shows reduced Cmax and enhanced-AUC. The observed pharmacokinetics data after
phase-High performance
oral administration of rifampicin along with piperine indicate significant enhancement in Cmax and AUC and thus
liquid chromatography
bioavailability. The bioavailability of isoniazid was reduced when it was co-administered with piperine, it could be
(RP-HPLC).
due to delay in gastric emptying time.

INTRODUCTION health public problem and is the single most deadly infectious
Rifampicin (RIF) (Figure 1), a complex semi- disease. It kills approximately two million people each year
synthetic macro cyclic antibiotic derived from Streptomyces (Gallieni et al., 1999). RIF is chemically (12Z, 14E, 24E)-
mediterranei, is a member of the RIF class of antibiotics (2S, 16S, 17S, 18R, 19R, 20R, 21S, 22R, 23S)-1,2-dihydro-5,
used for the treatment of tuberculosis and other infectious 6, 9, 17, 19-pentahydroxy, 23-methoxy-2, 4, 12, 16, 18, 20,
diseases (Maggi et al., 1966). It is categorized as one of the 22-heptamethyl-8-(4-methylpiperazin-1 yliminomethyl)-1,
first line antituberculous agent. Tuberculosis remains a major 11-dioxo 2, 7 (epoxypentadeca-1, 11, 13-trienimino) naphtha
[2,1-b] furan-21-yl acetate (Maryadele, 2006). It is official
in Indian Pharmacopoeia (IP, 2010), British Pharmacopoeia
Corresponding Author
* (BP, 2010) and United State Pharmacopoeia (USP, 2005). The
Amit Singh, School of Pharmacy, Monad University, Hapur, Uttar IP, BP and USP describe liquid chromatography and visible-
Pradesh, 245101, India. E-mail: amit21may @ rediffmail.com spectrophotometry method for its estimation. Literature

© 2018 Amit Singh et al. This is an open access article distributed under the terms of the Creative Commons Attribution License -NonCommercial- ShareAlikeUn-
ported License (http://creativecommons.org/licenses/by-nc-sa/3.0/).
 Singh et al. / Journal of Applied Pharmaceutical Science 8 (03); 2018: 072-081 073

survey reveals high performance liquid chromatography Piperine (PIP) (Figure 3) was discovered by Hans
(HPLC) (Panchagnula et al., 1999), high performance thin Christian Qrsted in 1819. He isolated it from the fruits of Piper
layer chromatography (HPTLC) (Shishoo et al., 2001) and nigrum, the source plant of both the black and white pepper grains.
visible spectrophotometry (Begum et al., 2013) methods Piperine is an alkaloid found naturally in plants belonging to the
for determination of RIF in pharmaceutical dosage forms as Piperaceae family, such as Piper nigrum L, commonly known as
well as in biological fluids. Literature survey also reveals black pepper and Piper longum L, known as long pepper.
spectrophotometric (Khuhawar and Rind, 1998), reverse phase
high performance liquid chromatography (RP-HPLC) (Calleri
et al. 2002) and visible spectrophotometry (Manna et al., 2000;
Ali et al., 2007) methods for determination of RIF within
combination drug.

Fig. 3: Chemical structure of piperine.

PIP is chemically 1-5-(1,3-benzodioxol-5-yl)-1-oxo–

2,4–pentadienyl piperidine (Sweetman 2005) is a natural alkaloids
use as bioenhancer (Atal et al., 1985). PIP is official in IP (2010),
describes liquid chromatography method for its estimation (Wood
et al., 1988). Literature survey also reveals HPLC (Nagappan et
al., 2009), UV-spectrophotometry (Gupta and Jain, 2011) and
HPTLC (Hamrapurkar et al., 2011; Shanmugasundram et al.,
2008) method for determination of PIP. Literature survey also
Fig. 1: Chemical structure of rifampicin.
reveals HPLC (Pattanaik et al., 2009) method for determination of
PIP with other drug combination. The combined dosages forms of
RIF and PIP along with INH are available in the market and used
as anti-tubular drugs.
Bio-enhancer is such compounds simply enhance the
bioavailability and bio-efficacy of particular drugs with which they
are combined without any pharmacological activity of their own
at the same dose level. PIP has increased the oral bioavailability
of several drugs which includes antitubercular, antileprotic,
antibiotics, NSAIDS, cardiovascular agents and central nervous
agent (Jaakiramnan and Manavalan 2008; Hiwale et al., 2002).
Fig. 2: Chemical structure of isoniazid. The aim of present method was to develop and validate
the simple, fast, sensitive and precise RP-HPLC method for the
Isoniazid (INH) (Figure 2), the hydrazide of assay of RIF, INH and PIP and also study the effect of PIP on
the bioavailability of RIF and INH when accessed in Swiss albino
isonicotinic acid is a synthetic analog of pyridoxine (Harvey
rats.
and Champe, 2000). It is the first line antitubercular medication
that never used on its own to treat active tuberculosis because MATERIALS AND METHODS
resistance quickly develops (Shinkich, 2007). Thus, it is widely
used together with RIF, ethambutol and pyrazinamide among Materials
others, for the chemotherapy of tuberculosis. Several methods The standard API of RIF and INH was received from
for the determination and quantitation of isoniazid have been ‘Exim’ Pharm International, Mumbai, India. PIP was obtained from
described. These include H-point standard addition method ‘Tulsi Bioscience’, Gujarat, India. Sodium carboxymethylcellulose,
(Safari et al., 2007), selective adsorption using a piezoelectric diethyl ether, EDTA, dextrose, chloroform of analytical grade was
sensor (Yao et al. 1999), voltametric method (Wahdan, purchased from Qualigens (Fischer), Mumbai, India. Methanol
2005), amperometric method (Quintino and Angnes, 2006), and potassium dihydrogen orthophosphate of HPLC grade were
chromatographic methods (HPLC, GC and HPTLC) (Moussa, procured from ‘Exim’ Pharm International, Mumbai, India.
2005; Carlina et al., 1998; Khuhawara and Zardari, 2008), Ethylene diamine tetra acetic acid (EDTA) obtained from ‘Sigma
titrimetric methods (Tatarczak et al., 2005), chemiluminisence St. Lowis’, MO, USA. Acetonitrile and dichloromethane were
(Juan et al., 2004) and UV-spectrometric (Nagendra et al., purchased from ‘Merck’, India. HPLC grade water was obtained
2002). from Millipore water purification system, USA.
074 Singh et al. / Journal of Applied Pharmaceutical Science 8 (03); 2018: 072-081

Table 1: Quality control samples concentration of rifampicin, isoniazid and piperine.

Rifampicin Isoniazid Piperine

Standard Reference Reference Reference

Strength Dilution Strength Strength
stock solution stock solution Dilution factor stock solution Dilution factor
(µg/mL) factor (µg/mL) (µg/mL)
(mg/mL) (mg/mL) (mg/mL)
QC STD 1 0.4 1 2500 0.4 1 2512.5 3 1 333.33
QC STD 2 250 1 4 75 1 13.4 250 1 4
QC STD 3 500 1 2 150 1 6.7 500 1 2

*QC STD- Quality Control Standard.

Apparatus pellet diet (Ashirwad Industries, Chandigarh, India) and water

RP-HPLC determinations were performed on ‘Shimadzu Ad libidum during the course of experimentation. Light cycle
LC 2010-HT’ with UV detector and ‘Shimadzu LC Solution was automatically controlled (on at 7 am and off at 7 pm) i.e. 12
Software’ 2010, equipped with auto sampler injector and a digital hr light-dark cycle. The room temperature and relative humidity
column oven. All weights were taken on electronic balance was maintained at 24 ± 2oC and 55 ± 10% respectively, that
(Mettler Toledo, Switzerland) and others solvent evaporator was maintained before the experimentation. After 3-5 days
(Thermo Electron Corporation, Milford, MA), centrifuge (Sigma acclimatization in animal house, rat was taken for experiment
St. Lowis MO, USA), sonicator (Banoelin Electronics, Berlin), purpose. All animals were exposed only once to every experiment.
digital pH meter (OAKTON) and vacuum filter (Millipore, USA)
were used. Dosing solutions
Dose for oral drug administration was prepared by
Quality control sample transferring accurately weighed quantity in 0.2% (w/v) solution of
The precision and accuracy parameters were ascertained sodium carboxymethyl cellulose, (Table 2).
in 3 different quality control samples, Table 1, ICH Q2R (1).
Table 2: Detail of drug dose given to rat.
LQC (Lower quality control)
Drug Dose
The lower quality control samples were prepared with
RIF- 0.4 µg/mL, INH- 0.4 µg/mL and PIP- 3 µg/mL. Rifampicin 10 mg/kg
Isoniazid 5 mg/kg
MQC (Middle quality control) Rifampicin + Isoniazid 10 mg/kg + 5 mg/kg
The middle quality control samples of RIF- 250 µg/mL, Rifampicin + Piperine 10 mg/kg + 1 mg/kg
INH- 75 µg/mL and PIP- 250 µg/mL was prepared. Isoniazid + Piperine 5 mg/kg + 1 mg/kg
Rifampicin + Isoniazid + Piperine 10 mg/kg + 5 mg/kg + 1 mg/kg
HQC (Higher quality control)
Higher quality control samples were prepared by Administration route
RIF- 500 µg/mL, INH- 150 µg/mL and PIP- 500 µg/mL of Drug was administered via oral route with the help of
concentrations. rat cannula and syringe, corresponding to the body weight of
Chromatographic conditions respective animals.
Chromatographic separation was carried out at 40°C on Test groups
a column Zorbax Eclipse XDP C18 (150 mm × 4.6 mm × 5 µm, GL
In the presence study six groups were selected having
Sciences, Inc. USA). The RIF, INH and PIP analyte were separated
six rats in each group.
with a mobile phase consisting of 20 mM potassium dihydrogen
orthophosphate (KH2PO4, pH = 4.5) and acetonitrile at 30:70 v/v Group I: Rifampicin
ratio. The flow rate was 0.8 mL/min. and the eluted analyte was Group II: Isoniazid
monitored by UV detection at 270 nm. Group III: Rifampicin + isoniazid
Group IV: Rifampicin + piperine
Animals Group V: Isoniazid + piperine
The in vivo study, on animal, was based on ‘Breeding Group VI: Rifampicin + isoniazid + piperine
of and Experiments on Animals (Control and Supervision)
Sample collection and handling
Rules 1998’ as amended by Government of India, Ministry of
Environment and Forests (CPCSEA), with permission number Blood was obtained by excising retro-orbital plexus
1149/ac/07/CPCSEA by Institutional Animal Ethical Committee under anesthesia with diethyl ether at the time interval of 30 min,
(IAEC). 1hr 30 min, 2 hrs, 4 hrs, 6 hrs, 9 hrs and 12 hrs. 10% EDTA solution
Albino Swiss rat (both sex), 9-14 weeks of age weight (100 µL per tube) was used as an anticoagulant. Intraperitoneal
range 200 to 280g were taken as per demand after the prior injections of dextrose (250 μL) were given to rats after collection
permission of the animal ethical committee. They were given of each blood sample to minimize changes in volume of the central
 Singh et al. / Journal of Applied Pharmaceutical Science 8 (03); 2018: 072-081 075

compartment. Plasma was obtained after centrifugation of blood of chemical studies that may or may not be distinguished from
samples at 5000 rpm for 10 min. each other. If the response is distinguished from all other response,
the method is said to be selective. Three different drug free plasma
Recovery of drug from the plasma matrix samples obtained from apparently selected animal rat were used
A fixed amount of plasma (250 µL) was transferred in to evaluate the selectivity of the developed method. This was done
test tube and 4 mL chloroform was added to them. Each sample by investigating the potential interference of blank plasma peak in
was vortexed on vortex mixer for 2 min. These extracted samples the drug peak area.
were centrifuged at 3000 rpm for 10 min, finally organic layer
collected in pre-labeled test tubes. These tubes were evaporated to Accuracy and recovery
dryness by using solvent evaporator. The accuracy of an analytical method is the extent to
which test results generated by the method and the true value
Analysis of samples agree. Accuracy can also described as the closeness of agreement
Residue left in each tube was reconstituted with mobile between the value that is adopted, either as a conventional, true or
phase which was prepared in degassed mixture of buffer and accepted reference value and the value found.
acetonitrile in the ratio of 30:70 (v/v). The sample was mixed by The accuracy of the method was determined by selecting
vortex shaker for a minute. Then it was filtered through 0.22 µm three (3) known concentrations of each analyte in plasma in
syringe filters. All the samples were analyzed by using HPLC the linearity range. The % recovery within day (intraday) and
apparatus with UV detection. interday, with standard deviations (±SD) was calculated to know
the accuracy of method.
Data analysis
After oral administration, the statistical analysis Precision and reproducibility
of observed pharmacokinetic parameters was determined by The precision of a method is the extent to which the
performing unpaired t-test (Graph Pad, Instat 3.0) to determine the individual test results of multiple injections of a series of standard
level of significance between two groups. P < 0.05 was considered agree. The measured standard deviation can be subdivided into 3
as the level of significance. categories: repeatability, intermediate precision and reproducibility.
Repeatability of the method was determined by
Pharmacokinetics calculating % RSD of the repeated six-determinates of same
Concentration-time curve was established for RIF, INH, 100% concentration of analyte drug in plasma. The intermediate
INH + RIF, RIF + PIP, INH + PIP and INH + RIF + PIP from precision was determined by selecting six different drug plasma
the treated rat (group I to VI), and used for the determination of samples by another analyst. The precision of the method was
pharmacokinetic parameters such as peak plasma concentration calculated by taking samples (6 replicates each in 3 sets) on the
(Cmax), peak time (Tmax), extent of absorption (AUC), half-life same day and on another day by calculating % bias from nominal
(t1/2), and elimination rate constant (Kel) were determined by using concentrations (quality control samples).
software ‘PK Solution’, a non-compartmental analysis. % Bias = [(mean measured conc. − nominal conc.)/
nominal conc.] × 100.
METHOD VALIDATION
The proposed method was validated according to the Sensitivity
ICH guidelines (ICH Q2A 1995 and ICH Q2B 1996), in terms of
linearity, specificity, suitability, accuracy, precision and stability. Limit of detection
The limit of detection is the point at which a measured
Linearity and calibration curve value is larger than the uncertainty associated with it. It is the lowest
The linearity of the calibration curve was determined by concentration of analyte in a sample that can be detected but not
regression equation and correlation coefficient (r2). The calibration necessarily quantified. LOD is calculated by measuring signal-to-
curve was constructed by plotting curve between drug concentration noise (S/N) of the baseline and multiplying this value by 3.
in plasma and average peak area of plasma samples (n = 3).
Limit of quantitation
Range The limit of quantitation is the minimum injected amount
The range of an analytical method is the interval that produces quantitative measurements in the target matrix
between the upper and lower levels (including these levels) that with acceptable precision in chromatography, typically requiring
have been demonstrated to be determined with precision, accuracy peak heights 10 times higher than the baseline noise i.e. LOQ is
and linearity using the method as written. The range is normally determinate by measuring signal-to-noise (S/N) and multiplying
expressed in the same units as the results (e.g., percentage, parts this value by 10.
per million) obtained by the analytical method.
Stability
Selectivity and specificity Many solutes readily decompose in plasma prior
The term specific generally refers to a method that to chromatographic investigations, for example, during the
produces a response for a single analyte only, while the term preparation of the sample solutions, extraction, cleanup, phase
selective refers to a method that provides response for a number transfer or storage of prepared vials (in refrigerators or in an
076 Singh et al. / Journal of Applied Pharmaceutical Science 8 (03); 2018: 072-081

automatic sampler). 16000000  

The stability of analyte in plasma was investigated 14000000
y = 21609x + 16628
R² = 0.9998
under the short-term and intermediate conditions. The short-term 12000000
stability of analyte was investigated immediately and after 24 10000000

Peak  Area
hrs at room and temperature at 10°C. The intermediate study was 8000000
planned for 1 month at low temperature. These conditions were; 6000000
Peak   Area
Linear (Peak   Area)
(a) 24 hr storage of drug-plasma sample at room 4000000
temperature (short term) 2000000
(b) 24 hr storage of drug-plasma sample at 10°C in auto 0
sampler (short term) 0 200 400 600 800

(c) 1 month storage of drug-plasma sample at −80°C and Concentration (µg/ml)

(intermediate)
(d) 3 consecutive freeze-thaw cycles of drug-plasma Fig. 4: Calibration plot of rifampicin.
sample from −80°C to room temperature (intermediate).
After specified storage conditions, drug-plasma samples
16000000
were analyzed by HPLC. The intra-assay and inter-assay accuracy
14000000 y = 23801x ‐ 45739
(% recovery) of the method was determined at zero time and in 12000000 R² = 0.9998
successive intervals from mean measured concentrations and 10000000

Peak       Area
nominal concentrations. 8000000 Series1
6000000 Linear (Series1)
RESULTS AND DISCUSSION 4000000
2000000
In the present study, the effect of PIP on pharmacokinetics
0
of RIF and INH was explored. The study also explores the new liquid ‐2000000 0 200 400 600 800
chromatographic method to analyze the RIF, INH, and PIP in plasma. Concentration (µg/ml)
 
Due to better separation parameters of RP-HPLC
technique, it has been frequently used in the analysis of Fig. 5: Calibration plot of isoniazid.
biologically samples. To achieve the high assay value of drug the
chromatographic condition were optimized in terms of mobile
phase ratio, column temperature, flow rate and pH of inorganic 900000
mobile phase. As expected an increase of organic mobile phase 800000 y = 1301x ‐ 1392.5
R² = 0.9996
from 10-25% resulted in decrease in retention time significantly 700000
600000
and simultaneous increase in peak height. The temperature
Peak  Area

500000
column in the range of 30-40°C slightly influences retention 400000 Series1
parameters. Based on the results described above, we selected 300000 Linear (Series1)
chromatographic condition as given in 2.4. 200000
100000
Analytical method 0
0 100 200 300 400 500 600 700

Linearity and range Concentration (µg/ml)

 
The calibration curve of RIF (Figure 4), INH (Figure
5), and PIP (Figure 6), were found satisfactorily in the range of Fig. 6: Calibration plot of piperine.
0.156-640 μg/mL. The linear regression equation for the analyte
were found Y = 21609X + 16628, Y = 23801X − 45739 and Y Accuracy and recovery
= 21609X + 16628. The linear regression data for above analyte Accuracy was determined by comparing the calculated
were found good because of showing high linear regression concentration of the sample with the true concentration of
coefficient (r2) that were the more than 0.999, proves the linearity rifampicin, isoniazid and piperine. The accuracy pertains to the
of calibration curve over the high range, (the regression coefficient extraction efficiency within the limit of variability. The accuracy
was demonstrated fit for purpose). of the method within inter and intraday was in ranged from 85 to
92.20%, that showed great back-calculation within the range (80-
Selectivity and specificity 120% according to ICH guidelines), Table 3a and 3b.
The developed method was found to specific because
none of the plasma components was interfered with the Precision and reproducibility
chromatograph of RIF, INH or PIP. It proves the specificity of the Precision and reproducibility was given as inter and intra-
developed method. day variations analyzed by three different concentration of rifampicin,
In an another test the separated and eluted sharp and isoniazid and piperine. The results were within the specified limit i.e.,
symmetric peaks of RIF, INH and PIP was found with an average variation in recovery was around ±15%, that proves the considerable
retention time of 7.59, 5.99 and 11.25 min. respectively. That degree of precision and reproducibility, Table 4.
proved the selectivity of the developed method.
 Singh et al. / Journal of Applied Pharmaceutical Science 8 (03); 2018: 072-081 077

Table 3a: Intra-day accuracy data of rifampicin, isoniazid and piperine. Table 3b: Inter day accuracy data of rifampicin, isoniazid and piperine.

Nominal †
Intra-day accuracy % Accuracy Nominal Inter day accuracy % Accuracy
Drug conc. Drug conc.
(µg/mL) Set 1 Set 2 Set 3 Average (µg/mL) Set 1 Set 2 Set 3 Average

0.4 0.35 ± 0.03 0.33 ± 0.02 0.37 ± 0.04 87.50 0.4 0.33 ± 0.02 0.35 ± 0.01 0.34 ± 0.02 85.00
RIF 250 221 ± 4.65 235 ± 6.93 229 ± 5.34 91.30 RIF 250 225 ± 5.94 231 ± 6.21 230 ± 6.32 91.63
500 471 ± 9.03 463 ± 10.84 449 ± 10.44 92.20 500 462 ± 10.54 452 ± 11.73 443 ± 10.52 90.46
0.4 0.37 ± 0.01 0.39 ± 0.01 0.34 ± 0.02 90.66 0.4 0.34 ± 0.01 0.35 ± 0.01 0.33 ± 0.01 85.16
INH 75 62 ± 2.13 69 ± 2.54 64 ± 2.46 86.65 INH 75 61 ± 2.79 65 ± 2.86 69 ± 3.14 86.65
150 141 ± 5.33 121 ± 5.87 128 ± 6.12 86.66 150 133 ± 6.15 129 ± 6.91 121 ± 6.88 85.00
3 2.88 ± 0.12 2.76 ± 0.15 2.53 ± 0.18 90.77 3 2.74 ± 0.17 2.79 ± 0.23 2.57 ± 0.19 91.90
PIP 250 236 ± 7.30 227 ± 7.15 223 ± 7.52 91.46 PIP 250 229 ± 7.97 221 ± 8.20 219 ± 8.11 89.20
500 458 ± 10.43 473 ± 12.22 453 ± 11.36 92.20 500 447 ± 11.31 463 ± 13.20 449 ± 14.3 89.60

Average of three (n = 3), RIF- Rifampicin, INH- Isoniazid, PIP- Piperine.

† †
Average of three (n = 3), RIF- Rifampicin, INH- Isoniazid, PIP- Piperine.

Table 4: Inter and intra-day precision data of rifampicin, isoniazid and piperine.

Intra-day precision Inter day precision

Nominal conc.
Drug *Percentage variation % Precision Percentage variation % Precision
(µg/mL)
Set 1 Set 2 Set 3 (†Average) Set 1 Set 2 Set 3 (†Average)

0.4 8.57 6.06 9.18 7.93% 4.24 2.85 6.17 4.42%

RIF 250 2.10 2.94 2.33 2.45% 2.64 2.68 2.74 2.68%
500 1.90 2.30 2.32 2.16% 2.28 2.59 2.37 2.41%
0.4 2.75 4.60 3.82 3.72% 2.90 3.40 3.31 3.20%
INH 75 3.43 3.60 3.84 3.62% 4.50 4.40 4.50 4.41%
150 3.78 4.85 4.78 4.50% 4.62 5.35 5.68 5.20%
3 4.00 5.43 7.10 5.51% 6.20 8.20 7.30 7.20%
PIP 250 3.09 3.10 3.37 3.18% 3.48 3.71 3.70 3.63%
500 2.27 2.58 2.50 2.45% 2.53 2.86 3.18 2.85%
†
Average of three (n = 3), Percentage variation- It is based on difference between standard nominal percentage conc. (100%) and test sample (set 1, 2 and 3), RIF- Rifampicin,
*

INH- Isoniazid, PIP- Piperine.

Sensitivity Table 5: Limit of detection and limit of quantitation of rifampicin, isoniazid

and piperine.
The sensitivity of the analytical technique was expressed
as the limit of quantification, which is the minimum concentration Drug LOD (µg/mL) LOQ (µg/mL)
of drug that can be quantitatively determined with a peak height to RIF 0.852 2.500
base line ratio of at least 10:1, and the limit of detection (LOD) as INH 0.689 2.102
peak height to base line ratio of 3:1, Table 7. It concludes that the PIP 0.756 2.224
developed method was sufficient sensitive to identify and quantify
RIF- Rifampicin, INH- Isoniazid, PIP- Piperine.
the drug rifampicin, isoniazid and piperine.

Stability studies Application of method in study of pharmacokinetics

The stability of analyte in plasma was investigated by The above developed HPLC method was utilized to
LQC, MQC and HQC samples. The recovery of the analyte at time study the effect of PIP on pharmacokinetics of RIF and INH.
zero (initial concentration) was assumed 100%. The stability of In this view, six groups of albino rat, each having six rats, was
analyte RIF, INH and PIP in ex-vivo at 0, 24 hr and 10°C, −80°C taken to know the effect of PIP on drugs with reference to
in plasma was tested. Under room temperature and at auto-sample pharmacokinetical parameters. The drugs were given to each
site the residual percentage of analyte RIF, INH and PIP were not
group of animal as given in Table 2. For validation purposes blank
more than 15% (i.e. % recovery was more than 85%) indicating no
plasma were collected before the dose administration.
stability problem, Table 5a.
In the intermediate stability the results corresponds The different pharmacokinetic parameters of RIF and
to those obtained after zero time and 1 month, the recovery INH were given in Table 7 that tells about the high peak plasma
percentage was not less than 85% (i.e. residual percentage not concentration (Cmax), peak time (Tmax), extent of absorption (AUC),
more than 15%) proves stability of the analyte drug RIF, INH and half-life (t1/2), and low elimination rate constant (Kel) of RIF and
PIP in plasma, Table 6b. INH respectively.
078 Singh et al. / Journal of Applied Pharmaceutical Science 8 (03); 2018: 072-081

Table 6a: Stability studies of rifampicin, isoniazid and piperine at 24h room temperature and storage at 10°C in auto sampler.

Nominal conc.
†
Recovery (µg) after storage at room temperature †
Recovery (µg) after storage in auto-sampler at 10°C
Drug
(µg/mL) 0 hr 24 hr % Stability†† 0 hr 24 hr % Stability††
0.4 0.35 ± 0.03 0.33 ± 0.02 91.42% 0.35 ± 0.03 0.31 ± 0.02 88.50%
RIF 250 228.30 ± 4.65 225 ± 6.93 98.50% 228.30 ± 4.65 215 ± 5.94 94.17%
500 461 ± 9.03 456 ±10.84 98.91% 461 ± 9.03 453 ± 10.54 98.26%
0.4 0.37 ± 0.01 0.36 ± 0.02 92.20% 0.37 ± 0.01 0.36 ± 0.01 98.07%
INH 75 65 ± 2.13 62 ± 2.54 95.30% 65 ± 2.13 61 ± 2.79 93.84%
150 130 ± 5.33 125 ± 5.87 96.15% 130 ± 5.33 126 ± 6.15 96.92%
3 2.72 ± 0.12 2.65 ± 0.15 97.40% 2.72 ± 0.12 2.67 ± 0.17 98.16%
PIP 250 228.60 ± 7.30 227 ± 7.15 99.30% 228.60 ± 7.30 221 ± 7.97 96.6%
500 461 ± 10.43 457 ± 12.22 99.2% 461 ± 10.43 455 ± 11.31 98.69%
†
Average of three (n = 3), ††After 24 hr of time % stability, RIF- Rifampicin, INH- Isoniazid, PIP- Piperine. Note: At ‘0’ hr (initial concentration) the recovery was
assumed to be 100%.

Table 6b: Stability studies of rifampicin, isoniazid and piperine when stored at −80°C.

Nominal conc. †Recovery (µg) after storage (−80°C)

Drug
(µg/mL) 0 month 1 month % Stability††
0.4 0.35 ± 0.03 0.33 ± 0.02 94.20%
RIF 250 228.30 ± 4.65 227 ± 6.93 99.40%
500 461 ± 9.03 456 ± 10.84 98.91%
0.4 0.37 ± 0.01 0.36 ± 0.02 97.70%
INH 75 65 ± 2.13 60 ± 2.54 92.30%
150 130 ± 5.33 121 ± 5.87 93.00%
3 2.72 ± 0.12 2.65 ± 0.15 97.40%
PIP 250 228.60 ± 7.30 227 ± 7.15 99.30%
500 461 ± 10.43 453 ± 12.22 98.20%
†
Average of three (n = 3), ††After one month % stability, RIF- Rifampicin, INH- Isoniazid, PIP- Piperine. Note: At ‘0’ month (initial concentration) the recovery was
assumed to be 100%.

Table 6c: Stability studies of rifampicin, isoniazid and piperine after 3 freeze-thaw cycles when stored at −80oC.

Nominal conc. †Recovery (µg) after freeze-thaw cycles

Drug
(µg/mL) Cycle 0 Cycle 1 Cycle 2 Cycle 3 % ¥Stability
0.4 0.35 ± 0.03 0.33 ± 0.02 0.31 ± 0.01 0.32 ± 0.02 91.40%
RIF 250 228.30 ± 4.65 225 ± 5.94 221 ± 6.21 222 ± 6.32 97.53%
500 461 ± 9.03 459 ± 10.54 452 ± 11.73 453 ± 10.52 98.62%
0.4 0.37 ± 0.01 0.34 ± 0.01 0.35 ± 0.01 0.33 ± 0.01 93.84%
INH 75 65 ± 2.13 61 ± 2.79 62 ± 2.86 59 ± 3.14 96.41%
150 130 ± 5.33 123 ± 6.15 129 ± 6.91 121 ± 6.88 95.64%
3 2.72 ± 0.12 2.71 ± 0.17 2.68 ± 0.23 2.57 ± 0.19 97.50%
PIP 250 228.60 ± 7.30 225 ± 7.97 221 ± 8.20 225 ± 8.11 97.80%
500 461 ± 10.43 457 ± 11.31 455 ± 13.28 449 ± 14.30 98.40%
†
Average of three (n = 3), ¥Average of cycle 0, 1, 2 and 3, RIF- Rifampicin, INH- Isoniazid, PIP- Piperine. Note: At ‘0’ cycle (initial concentration) the recovery was
assumed to be 100%.

Pharmacokinetics of rifampicin and isoniazid (combined (Figure9). The difference in AUC was found to be statistically
administration-dose) significant (p < 0.001) when RIF was administered alone or in
When the RIF and INH was given as combined combination with INH. Further the Cmax was also found to be
administration-dose, the mean plasma concentrations of RIF with reduced when it was administered in combination with INH.
INH were lower as compared to RIF alone, at all time points Relative bioavailability of RIF was found to be 67.19%, Table 8.
 Singh et al. / Journal of Applied Pharmaceutical Science 8 (03); 2018: 072-081 079

Table 7: Pharmacokinetics parameters of rifampicin and isoniazid (combined administration-dose).

††
Rifampicin ††
Isoniazid
Pharmacokinetic parameters
Alone Combined administered dose Alone Combined administered dose
Cmax (µg/mL) 8.85 ± 1.65 6.49 ± 1.75 8.05 ± 4.75 8.11 ± 1.82
Biological half life (h) 2.80 ± 0.42 2.04 ± 0.42 1.04 ± 0.22 1.02 ± 0.18
†
Area under curve (AUC0 to ∞) 40.36 ± 3.59 27.12 ± 4.59 24.77 ± 3.59 27.03 ± 3.88
Tmax (h) 2.12 ± 0.48 2.20 ± 0.63 0.50 ± 0.04 0.50 ± 0.08
Kel (h−1) 0.24 ± 0.02 0.33 ± 0.04 0.66 ± 0.07 0.67 ± 0.09
†
Unit of area under curve (µg.h/mL), Average of six (n = 6).
††

Table 8: Pharmacokinetics parameters of rifampicin and piperine (combined Table 9: Pharmacokinetics parameters of isoniazid and piperine (combined
administration dose). administration dose).

Pharmacokinetics parameters Rifampicin

††
Rifampicin + Piperine
††
Pharmacokinetics Parameters ††
Isoniazid ††
Isoniazid + Piperine
Cmax (µg/mL) 8.85 ± 1.75 13.63 ± 1.82 Cmax (µg/mL) 8.40 ± 0.47 5.33 ± 1.02
Biological half life (h) 2.80 ± 0.42 3.09 ± 0.28 Biological half life (h) 1.40 ± 0.38 1.20 ± 0.26
†
Area under curve (AUC0 to ∞) 40.36 ± 3.59 58.03 ± 3.88 Area under curve, (AUC0 to ∞) 24.77 ± 4.59 13.36 ± 3.78
Tmax (h) 2.20 ± 0.63 2.42 ± 0.78 Tmax (h) 0.50 ± 0.08 0.50 ± 0.05
Kel (h−1) 0.24 ± 0.02 0.23 ± 0.09 Kel (h−1) 0.66 ± 0.22 0.66 ± 0.09
†
Unit of area under curve (µg.h/mL), Average of six (n = 6). ††

Table 10: Pharmacokinetics parameters of rifampicin, isoniazid and piperine (combined administration dose).

††
Isoniazid Rifampicin
††

Pharmacokinetics parameters
Alone Combined administered dose Alone Combined administered dose
Cmax (µg/mL) 8.05 ± 4.75 5.35 ± 2.75 8.85 ± 1.65 13.63 ± 0.82
Biological half life (h) 1.04 ± 0.22 1.05 ± 0.42 2.80 ± 0.42 3.09 ± 0.28
†
Area under curve (AUC0-∞) 24.77 ± 3.59 13.36 ± 2.59 40.36 ± 3.59 58.03 ± 3.78
Tmax (h) 0.50 ± 0.04 2.20 ± 0.63 2.12 ± 0.48 2.42 ± 0.78
Kel (h−1) 0.66 ± 0.07 0.66 ± 0.12 0.24 ± 0.02 0.22 ± 0.09
†
Unit of area under curve (µg.h/mL), ††Average of six (n = 6).

Pharmacokinetics of rifampicin and piperine (combined  

administration-dose) 10
Mean Plasma Conc. of RIF (µg/ml)

9
The mean plasma concentrations of RIF were found to 8 RIF
be higher with PIP as compared to the administration of RIF alone 7
6 AUC
(Figure 10). The difference was statistically significant at 0.5, 1, 1.5, 5 RIF= 40.36 µg.h/ml
2 and 4 hr. Table 9 compares various pharmacokinetic parameters 4
of RIF when it was administered alone and in combination with 3
2
PIP. Further Cmax of RIF was found to be significantly higher when 1
administered along with PIP (p < 0.001). Relative bioavailability 0
of INH was found to be 141.7% when it was co-administered with 0 2 4 6 8 10 12 14
Time (hrs)
PIP.

Pharmacokinetics of isoniazid and piperine (combined Fig. 7: Mean plasma concentration of rifampicin administered a dose of 10
mg/kg by oral route.
administration dose)
The mean plasma concentrations of INH was found to Pharmacokinetics of rifampicin, isoniazid and piperine
be lower with PIP as compared to the administration of INH alone (combined administration dose)
(Figure 12). The difference was statistically significant at 0.5, Plasma concentration was reduced when INH was
1, 1.5, 2 and 4 hr. Table 10, compares various pharmacokinetic administered in combination with PIP (p < 0.001). Further plasma
parameters of INH when it was administered alone and in concentration of RIF was enhanced when it was co-administered
combination with PIP. Further Cmax of INH was found to be with PIP (p < 0.001). However Cmax of RIF when administered
significantly lower when administered along with PIP (p < 0.001). with PIP was found to be significantly enhanced (p < 0.001) when
Relative bioavailability of INH was found to be 53.9% when it compare to RIF alone. Relative bioavailability of RIF was found
was co-administered with PIP. to be 141.7%. Cmax of INH was found to be reduced when it was
080 Singh et al. / Journal of Applied Pharmaceutical Science 8 (03); 2018: 072-081

co-administered with PIP (p < 0.001). Relative bioavailability of Pharmacokinetic profile indicates that the PIP enhanced
INH was observed to be 53.9% when it was co-administered with the rate and extent of absorption of RIF (P < 0.001). The observed
PIP, Table 11. pharmacokinetic data after oral administration of RIF along with
PIP indicate a significant enhancement in Cmax (8.85 ± 1.75 to
CONCLUSION
13.62 ± 1.82) and AUC (40.36 ± 3.59 to 58.03 ± 3.88). However,
In this study an effective RP-HPLC method was the plasma elimination half life were found to be similar which
developed for determination of RIF, INH and PIP with high is likely due to the reduced gastrointestinal absorption of RIF
accuracy and precision. Thus, it can be concluded that the proposed caused by INH and degradation of RIF and degradation of RIF in
method was sufficiently sensitive, reproducible and specific for
presence of INH. Unfortunately, the oral bioavailability of INH
analysis of RIF, INH and PIP in biological samples or plasma.
was reduced when it was co-administered with PIP. The observed
The proposed method was also validated by evaluating different
pharmacokinetic profile found to be reduced Cmax (8.4 ± 0.47 to
parameter according to ICH guidelines like specificity, sensitivity,
5.328 ± 113.36 ± 3.78.02) and AUC (24.77 ± 4.59 to 58.03 ±
accuracy, precision, and stability of the analyte.
3.78) and biological half life (1.04 ± 0.1 h). The possible reason
for decline may be the PIP antagonizes effects of acetylcholine on
isolated ileum result of delay in gastric empty rate and delay the
Isoniazid
9 exposure of INH to vast absorptive surface of the small intestine.
Mean Plasma conc. of Isoniazid

8
7
6 AUC 9 INH INH + PIP
5 INH= 24.77µg.h/ml

Mean Plasma conc. of Isoniazid

8
(µg/ml)

4
7
3
2 6
1 5 AUC

(µg/ml)
0 4 INH= 24.77 µg.h/m
ml
0 2 4 6 8 10 12 14 PIP= 13.36µg
INH+P g.h/ml
3
Time (h) 2
  1
0
0 2 4 6 8 10 12 14
Fig. 8: Mean plasma concentration of isoniazid administered a dose of 5 mg/
kg by oral route. Time (h)
 

  Fig. 11: Mean plasma concentration of isoniazid administered a dose of 5 mg/

9 kg and piperine 1 mg/kg by oral route (combined administration dose).
RIF                                   INH
8
Plasma Conc. (µg/ml)

7
16  
6
5 AUC 14 INH + PIP RIF + PIP
RIF= 27.12µg.h/ml
4 INH= 27.08µg.h/ml 12
Plasma conc. (µg/ml)

3 10
2
8 AUC
1 INH + PIP= 13.36 µg.h/ml
6
0 RIF + PIP= 58.03µg.h/ml
0 2 4 6 8 10 12 14 4
Time (h) 2
0
Fig. 9: Mean plasma concentration of rifampicin administered a dose of 10 0 2 4 6 8 10 12 14
mg/kg and isoniazid 5 mg/kg by oral route (combined administration dose). Time (h)

Fig. 12: Mean plasma concentration of rifampicin administered a dose of

16
10 mg/kg, isoniazid 5 mg/kg and piperine 1 mg/kg by oral route (combined
RIF RIF+PIP administration dose).
14
Mean of Plasma conc of RIF

12
10
DECLARATION OF INTEREST
8 AUC
(µg/ml)

6
RIF= 40.36µg.h/ml The authors confirm that this article contents and writing
RIF+PIP= 58.0µg.h/ml
4 of the paper has no conflicts of interest.
2
0 ACKNOWLEDGEMENTS
0 2 4 6 8 10 12 14
Time (h)
The authors would like to extend their sincere
 
appreciation to Sheetal Nagar, Teva API India Ltd. Ecotech – II,
Udyog Vihar, Greater Noida, Gautam Budh Nagar, Uttar Pradesh,
Fig. 10: Mean plasma concentration of rifampicin administered a dose of 10 India, 201 306.
mg/kg and piperine 1 mg/kg by oral route (combined administration dose).
 Singh et al. / Journal of Applied Pharmaceutical Science 8 (03); 2018: 072-081 081

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