5.1 introduction - shodhgangashodhganga.inflibnet.ac.in/bitstream/10603/11199/9/chapter - 5.pdf ·...

Chapter5 Rifaximin Introduction

128

5.1 Introduction

Rifaximin is (2S, 16Z, 18E, 20S, 21S, 22R, 23R, 24R, 25S, 26S, 27S, 28E)- 5,

6, 21, 23, 25 - pentahydroxy-27-methoxy-2, 4, 11, 16, 20, 22, 24, 26-octamethyl-2,7-

(epoxy pentadeca- [1, 11, 13] trienimino) benzofuro [4, 5-e]pyrido [1, 2-a]-

benzimidazole-1,15(2H)-dione,25-acetate. The empirical formula is C43H51N3O11

and its molecular weight is 785.89 (Fig-5.1).

Rifaximin Rifaximin-D6

Figure 5.1 Chemical structures of Rifaximin sodium (A),Rifaximin - d6 (B).

Rifaximin is a semisynthetic, rifamycin-based non-systemic antibiotic,

meaning that very little of the drug will pass the gastrointestinal wall into the

circulation as is common for other types of orally administered antibiotics. It is used

in the treatment of traveler's diarrhea and hepatic encephalopathy 1.Rifaximin is

poorly absorbed from the gastrointestinal tract. Although food significantly increases

the extent of absorption of rifaximin , as expressed by AUC, Cmax and urinary

excretion, systemic absorption of rifaximin was lower than 1% (based on urinary


129

excretion) in both the fasting state and when administred with in 30 minutes of a

high-fat breakfast 6,7

Several techniques for quantification of Rifaximin, such as U.V5

HPLC3,LC-MS/MS

2,4 have been reported .UV method involves a tedious extraction

procedure involving too many steps. This may result inaccurate and imprecise results.

Quantification of Rifaximin in pharmaceutical dosage forms by U.V6and

Quantification of Rifaximin in rat plasma 4,5

was developed. Only a few methods were

developed for quantification of Rifaximin in human plasma by using HPLC-MS3.

They have developed sensitive method observed with good leniearity between the

concentration range 0.5- 10 ng/ml. The extraction of drug and IS was achieved by

precipitation method which results less recovery, less precise results. Nowadays,

HPLC-MS has gained importance for the quantitative estimation of drugs in various

biological matrices including plasma, serum, urine, and ocular fluids, due to its high

sensitivity, selectivity and reproducibility.

The main goal of the proposed work is to prove highly sensitive, selective,

good linear, reproducible and simple extraction analytical method for quantification

of Rifaximinin plasma samples. On the other hand, comparison of drug with

deuterated internal standard for short run times and for less matrix effect which are

most useful in analysis by using HPLC-MS. The developed method could be

employed in the analysis of plasma samples in clinical / Pharmacokinetic study.


130

5.2 Experimental Investigations

5.2.1 Materials and reagents

Rifaximin was obtained from sigma Aldreich Bangalore, India. Rifaximin -D6

internal standard was obtained from TRC (Torrent Research Chemicals, Ontario)

Canada. Acetonitrile, Methanol, were Obtained from J.T.Baker, USA. Ammonium

formate, Formic acid, Methyl t-butyl ether, Dichloromethane, Orthophosphoric acid

were purchased from Merck Speciality Chemicals Ltd, Mumbai, India. Human

plasma was procured from Navazeevan Blood bank, Hyderabad. Millipore water was

used from MilliQ system.

5.2.2 Instrumentation and equipment Refer Chapter-3.2.2

5.2.3 Preparation of reagents and solvents Table 5.1 Preparation of reagents and solvents

Reagents and solvents preparation

50% Acetonitrile Mix 500 mL of Acetonitrile with 500 mL of water.

10mMAmmonium formate Dissolve 1.26 g of ammonium formate into 2 L of water.

0.1%Formic Acid Dilute 1 mL of formic acid to 1000 mL of water.

Orthophosphoric acid

solution Dilute 5 mL of orthophosphoric acid to 1000 mL of water.

Extraction solvent Mix 750 mL of methyl t-butyl ether with 250 mL of dichloromethane.

Mobile phase

Mix 10mM Ammonium formatepH 4.0 : Acetonitrile in the ratio of 20:80

and

Filter through 0.45 m filter

Reconstitution solution Mix 200 mL of 10 mM ammonium formate, pH 4.0 with 800 mL of

acetonitrile.

(Autosampler wash)

80% Acetonitrile Mix 800 mL of Acetonitrile with 200 mL of water.


131

5.2.4 Preparation of stock solution

Table 5.2 Preparation of stock solution

Name of the stock

solutions Concentration Volume (ml) Diluent

Rifaximin 100.0 g/ml 100 ml Methanol

Rifaximin D6 100.0 g/ml 100 ml Methanol

5.2.5 Preparation of standards and quality control (QC) Samples

Standard stock solutions of Rifaximin (100µg/mL), and the IS (100µg/mL)

master stock solutions were prepared in methanol. The IS spiking solutions (20

ng/mL) were prepared in 50% acetonitrile from IS stock solution. Standard stock

solutions and IS spiking solutions stored in refrigerator conditions 2-8 °C until

analysis. Standard stock solutions were added to drug-free human plasma to obtain

Rifaximin concentration levels of 20.0, 40.0, 200.0, 1000.0, 2000.0, 4000.0,

8000.0,12000.0, 16000.0 and 20000.0 pg/mL for analytical standards and 20.0, 60.0,

6000.0, 14000.0 pg/mL for Quality control standards and stored in a -30°C set point

freezer until analysis. Rinse all glassware with 0.1% formic acid, water and methanol

before dilutions. (All dilutions are made with plasma placed in ice/ water bath in the

absence of white light). The Aqueous standards were prepared in reconstitution

solution (10mM ammonium format (PH 4.0) and acetonitrile in the ratio of (20:80

v/v),) for validation exercises until analysis.


132

5.3 Method Development

The goal of this research is to develop and validate a simple, selective,

sensitive, rapid, rugged and reproducible assay method for the quantitative

determination of Rifaximin from plasma samples. In the way to develop a simple and

easy applicable method for Rifaximin assay in human plasma for pharmacokinetic

study, HPLC with MS/MS detection was selected as the method of choice.

Mass parameter Optimization, Chromatographic Optimization and Extraction

optimization to be optimized carefully to achieve the best results.

The MS optimization was performed by direct infusion of solutions of both

Rifaximin and Rifaximin D6 into the ESI source of the mass spectrometer. Other

parameters, such as the nebulizer and the heater gases and Declustering potential(DP),

Entrance potential(EP),Collision energy(CE) was optimized to obtain a better spray

shape, resulting in better ionization and droplet drying to form the protonated ionic

Rifaximin and Rifaximin D6 molecules.

A CAD product ion spectrum for Rifaximin and Rifaximin D6 yielded high-

abundance fragment ions of m/z(amu) 754.4 and m/z(amu) 760.5 respectively

(Fig.5.2,to Fig. 5.5).


133

Figure 5.2:Parent ion mass spectra (Q1) of Rifaximin


134

Figure 5.3:Product ion mass spectra (Q3) of Rifaximin


135

Figure 5.4:Parent ion mass spectra (Q1) of Rifaximin-D6


136

Figure 5.5:Product ion mass spectra (Q3) of Rifaximin-D6

Chromatographic conditions, especially, selection of column, composition and

nature of the mobile phase were optimized through several trials to achieve best

resolution and increase the signal of Rifaximin and Rifaximin D6. Separation was

tried using various combinations of mobile phase with variety of columns like YMC

Pack pro C18, RP-Amide, Ascentis Express RP-amide, X-Bridge, Discovery Cyano,


137

Kromasil 100- 5CN. After the MRM channels were tuned, the mobile phase was

changed from more aqueous phase to organic phase to obtain a fast and selective LC

method. A good separation and elution were achieved using 10 mM ammonium

formate(PH 4.0): acetonitrile (20:80 v/v) as the mobile phase, at a flow-rate of 0.3mL/

min and injection volume of 5 µl. Chromatographic analysis of the analyte and IS was

initiated under isocratic conditions with an aim to develop a simple separation process

with a short run time.

Extraction was performed by different extraction techniques like SPE, LLE,

Precipitation methods. Finally a simple LLE technique was selected in the extraction

of Rifaximin and Rifaximin D6 from the plasma samples.

Chromatographic conditions

An aliquot of the plasma extract (5 L) was injected into Zorbax SB C18, 4.6 x

75 mm, 3.5 µm column. The column was placed at a temperature (40oC). Both drug

and internal standard were eluted at 3.3 ± 0.2 min with a run time of 5 min between

injections. Separation and elution were achieved using 10 mM ammonium

formate(pH 4.5) :acetonitrile (20:80v/v) as the mobile phase, at a flow-rate of 0.3

mL/min.

Sample preparation

Liquid-liquid extraction (LLE) was used to isolate Rifaximin and Rifaximin

D6 from human plasma. 50 µL of Rifaximin D6 (20.0 ng/ mL) and 400 µL respective

plasma concentrations were added into polypropylene tubes and vortexed briefly. This

was followed by addition of 100 µL of orthophosphoric acid solution and 3.0 ml of


138

extraction solvent (methyl t-butyl ether : dichloromethane (75: 25) into each tube and

vortexed for 20 min. All the samples were centrifuged at 4000 rpm, 20°C for 10 min

and transferred the supernatant from each sample into respective polypropylene tubes.

Samples were evaporated to dryness under nitrogen at 40°C. Finally samples were

reconstituted with 200 µL of reconstitution solution (10mM ammonium formate

pH(4.0) : acetonitrile (20:80 v/v)) and vortexed briefly. From this 5 µL of each

sample was injected into the HPLC system connected to the mass spectrometer.

Extraction was carried out under absence of white light.

Calibration curve parameters and regression model

The analytical curves of Rifaximin were constructed in the concentrations

ranging from 20-20000.0 pg/mL in human plasma. Calibration curves were obtained

by weighted linear regression (weighing factor: 1/x2). The ratio of Rifaximin peak

area to RifaximinD6 peak area was plotted against the ratio of Rifaximin

concentration in pg/mL. The fitness of calibration curve was confirmed by back-

calculating the concentrations of calibration standards.

Method Development Conclusion

The developed method is suitable for estimation of plasma concentrations for

Rifaximin as a single analytical run, in clinical samples from Pharmacokinetic studies.

This was followed by method validation.


139

5.4 Method Validation

The objective of the work is to validate specific HPLC- MS method for the

determination of Rifaximin in human plasma for clinical / Pharmacokinetic study.

Chromatography

Representative chromatograms of Plasma blank, blank +IS, LOQ, ULOQ,

LLOQC, LQC, MQC, HQC, Calibration curve are shown in Figure 5.6 to 5.14.

Figure 5.6 Chromatogram of Blank Human Plasma Sample


140

Figure 5.7 Chromatogram of Blank + IS


141

Figure 5.8: Chromatogram of LOQ Sample (Rifaximin & IS)


142

Figure 5.9 Chromatogram of ULOQ Sample (Rifaximin & IS)


143

Figure 5.10: Chromatogram of LLOQ Sample (Rifaximin & IS)


144

Figure 5.11: Chromatogram of LQC Sample (Rifaximin & IS)


145

Figure 5.12 Chromatogram of MQC Sample (Rifaximin & IS)


146

Figure 5.13: Chromatogram of HQC Sample (Rifaximin & IS)


147

Figure 5.14 Calibration Curve of Rifaximin


148

Blank Matrix Screening

During validation, blank plasma samples from 10 different lots were processed

according to the extraction procedure and evaluate the interference at the retention

times of analyte and internal standard. The 6 free interference lots were selected from

the 10 lots. Results are presented in table 5.3.

Table 5.3 Screening of Different batches of blank matrix for interference free

Rifaximin blank plasma (Human K2EDTA Plasma)

Matrix identification

Blank plasma Area

Analyte

(Rifaximin)

RT

Internal standard

RT

PL Blank-(K2EDTA-AP/2451/09/10) 0 0


PL Blank-(K2EDTA-AP/2453/09/10) 14 0








Blank+IS with PL Blank-(K2EDTA-

AP/2451/09/10) 0 262864

LOQ with PL Blank-(K2EDTA-

AP/3451/09/10) 2825 266426

Blank Matrix Specificity and Limit of Quantification

During specificity run, the LLOQ standard was prepared in one of the

screened blank plasma including the spiking of working range of internal standard.

Blank plasma samples from 10 different lots, 6 LLOQ standards were processed

according to the extraction procedure. The responses for the blank plasma from 10

different lots were compared to the LLOQ standard of the analyte and internal


149

standard. No significant response (≤20% for the analyte response and ≤5% of the

internal standard response) was observed at the retention times of the analyte or the

internal standard in blank plasma as compared to the LLOQ standard. Results are

presented in table 5.4.

The specificity experiment shall be considered for calculation of LOQ

experiment. Results are presented in table 5.5

Table 5.4 Specificity of Different batches of blank matrix (K2EDTA Plasma)

Matrix

Identification

LLOQ

Area

Internal

standard

(IS) area

Interference with

Analyte(% of

LLOQ Response)

Interference

with IS(% of IS

Response)

PL Blank-

(K2EDTA-

AP/2451/09/10) 2478 439772 0 0

PL Blank-

(K2EDTA-

AP/2452/09/10) 2825 412836 0 0

PL Blank-

(K2EDTA-

AP/2453/09/10) 2712 416511 0 0

PL Blank-

(K2EDTA-

AP/2454/09/10) 2599 399305 0 0

PL Blank-

(K2EDTA-

AP/2455/09/10) 2779 406926 0 0

PL Blank-

(K2EDTA-

AP/2456/09/10) 2576 418875 0 0

Acceptance criteria:

1. Analyte response should be ≤ 20% of LOQ Response in at least 75% of the

blank.

2. Internal standard response should be ≤5% of mean internal standard response

in at least 75% of the blank.


150

Table 5.5 Limit of Quantification for analyte (Rifaximin)

Matrix identification Blank plasma area at

Analyte RT

LLOQ

response

LLOQ S/N

RATIO

PL Blank-(K2EDTA-

AP/2451/09/10)

0 2478 18.7

0 2825 16.8

0 2712 18.9

0 2599 13.2

0 2779 23.5

0 2576 14.4

N 6 6 6

Mean 0 2662 17.6

LLOQ was spiked in PL Blank-(K2EDTA-AP/2451/09/10)


1. Mean S/N ratio of LLOQ should be ≥5.

2. S/N ratio is analyst software generated data.

Intra Batch Accuracy and precision

Intra batch accuracy and precision evaluation were assessed by analyzing 1

calibration curve and 6 replicate each of the LLOQ, LQC, MQC, HQC, from

precision and accuracy batch-1.

The Intra batch percentage of nominal concentrations for Rifaximin was

ranged between 95.70% and 108.00%.

The Intra batch percentage of coefficient of variation is 0.6% to 3.2% for

Rifaximin.

Results are presented in table 5.6


151

Table 5.6 Intra batch (Within-Batch) Accuracy and Precision for determination

of Rifaximin levels in human plasma

Analytical

Run ID

LLOQ

20.00 ng/ml

Low QC

60.00 ng/ml

Mid QC

6000.00 ng/ml

High QC

14000.00 ng/ml

Conc.

found

%

nominal

Conc.

found

%

nominal

Conc.

found

%

nominal

Conc.

found

%

nominal

P&A Batch 1

21.2 106.00 61.9 103.17 6135.2 102.25 13380.1 95.57 22.8 114.00 60.2 100.33 6011.1 100.19 13334.4 95.25 21.1 105.50 65.1 108.50 6181.9 103.03 13323.5 95.17

21 105.00 61.8 103.00 6164.1 102.74 13489.7 96.36 22 110.00 62.7 104.50 6209.6 103.49 13522.7 96.59

21.7 108.50 63.3 105.50 6122.7 102.05 13378.5 95.56

N 6

6

6

6

Mean 21.6 62.5 6137.4 13404.8

SD (±) 0.7 1.6 69.4 82.4

CV (%) 3.2 2.6 1.1 0.6

%Accuracy 108.00 104.20 102.30 95.70


1. % CV ≤ 15 % except LLOQ for which it is ≤ 20%.

2. Mean % Nominal (100±15% and for LLOQ 100±20%).

Inter Batch Accuracy and Precision

Inter batch accuracy and precision evaluation were assessed by analyzing 5

sets of calibration curves for Rifaximin and 5 sets of QC samples, 6 replicates each of

the LLOQ, LQC, MQC and HQC.

The inter batch percentage of nominal concentrations for Rifaximin was

ranged between 95.8% and 105.00%.

The Inter batch percentage of coefficient of variation is 2.2% to 6.4% for

Rifaximin.

Results are presented in table 5.7


152

Table 5.7 Inter batch (Between-Batch) Accuracy and Precision for determination

of Rifaximin levels in human plasma

Analytical

Run ID

LLOQ

20.00 ng/ml

Low QC

60.00 ng/ml

Mid QC

6000.00 ng/ml

High QC

14000.00 ng/ml

Conc.

found

%

nominal

Conc.

found

%

nominal

Conc.

found

%

nominal

Conc.

found

%

nominal

P&A

Batch 1

21.2 106.00 61.9 103.17 6135.2 102.25 13380.1 95.57 22.8 114.00 60.2 100.33 6011.1 100.19 13334.4 95.25 21.1 105.50 65.1 108.50 6181.9 103.03 13323.5 95.17

21 105.00 61.8 103.00 6164.1 102.74 13489.7 96.36 22 110.00 62.7 104.50 6209.6 103.49 13522.7 96.59

21.7 108.50 63.3 105.50 6122.7 102.05 13378.5 95.56

P&A

Batch 2

21.3 106.50 62 103.33 6053.6 100.89 13531.6 96.65 19.9 99.50 60.6 101.00 6042.7 100.71 13232.3 94.52 20.1 100.50 58.5 97.50 6133 102.22 13247.5 94.63 20.9 104.50 61.3 102.17 6121 102.02 13631.8 97.37 20.4 102.00 61.9 103.17 6261.5 104.36 13493.3 96.38

21 105.00 61.4 102.33 6182.4 103.04 13468 96.20

P&A

Batch 3

19.1 95.50 60.4 100.67 6094.5 101.58 13384.5 95.60 18.5 92.50 59 98.33 6072.8 101.21 13331.2 95.22 19.4 97.00 60.6 101.00 6049.3 100.82 13370.1 95.50 19.4 97.00 61 101.67 6177.9 102.97 13754.6 98.25 20.8 104.00 64.8 108.00 6064.5 101.08 13532.1 96.66 19.3 96.50 67.5 112.50 6055.9 100.93 13894.8 99.25

P&A

Batch 4

18.3 91.50 66.8 111.33 5556.2 92.60 13453.6 96.10 19.5 97.50 66.7 111.17 5622.2 93.70 13339.2 95.28 19.3 96.50 66.2 110.33 5828.8 97.15 13610.7 97.22 20.7 103.50 67.3 112.17 5792.4 96.54 13386.9 95.62 20.2 101.00 61.5 102.50 6455.3 107.59 13501.4 96.44 18.1 90.50 68.7 114.50 6447.4 107.46 13220.6 94.43

P&A

Batch 5

18.8 94.00 57.9 96.50 5819.5 96.99 12506.9 89.34 19.2 96.00 58.1 96.83 5960.9 99.35 12704.8 90.75 19.5 97.50 62.3 103.83 6001.3 100.02 12967.2 92.62 20.8 104.00 61.1 101.83 6113.2 101.89 13532.3 96.66 22.8 114.00 62.7 104.50 6177.3 102.96 13758.1 98.27 22.1 110.50 62.6 104.33 6496.1 108.27 13942 99.59

N 30

30

30

30

Mean 20.3 63 6080.1 13407.5 SD(±) 1.3 3.5 210.3 297.3

CV (%) 6.4 5.6 3.5 2.2

%Nominal 101.5 105 101.3 95.8

Acceptance criteria: Same as Table 5.6


153

Calibration Curve

Calibration curves are found to be consistently accurate and precise for

Rifaximin over 20.00 to 20000.00pg/ml for calibration range. The correlation

coefficient is greater than 0.9995 for Rifaximin. Back calculations were made from

the calibration curves to determine Rifaximin concentrations of each calibration

standard.

Results are presented in tables 5.8 & 5.9.

Table 5.8 Summary of calibration curve parameters for Rifaximin in human

plasma

Analytical Run ID slope intercept Coefficient of

regression (r2)

P&A Batch-1 0.0004707 0.0008451 0.9997

P&A Batch-2 0.0004798 0.001239 0.9996

P&A Batch-3 0.0004646 0.001559 0.9995

P&A Batch-4 0.0004842 0.001411 0.9997

P&A Batch-5 0.0004595 0.001569 0.9995

N 5 5 5

Mean 0.0004718 0.001325 0.9996

SD (±) 0.00001027 0.0002997 0.0001

CV (%) 2.2 22.6 0

1) Resp. = Slope * Conc. + Intercept


1. Coefficient of regression (r) ≥0.9980.


154

Table 5.9 Back-calculated standard concentrations from each calibration curve

for Rifaximin in human plasma.

Analytical Run

ID

Nominal Concentration (ng/ml)

CS1 CS2 CS3 CS4 CS5

20.00

pg/ml

40.00

pg/ml

200.00

pg/ml

1000.00

pg/ml

2000.00

pg/ml

P&A Batch-1 19.8 40.8 201 1009.3 1987

P&A Batch-2 19.9 40.1 207.7 996.5 1947

P&A Batch-3 20.4 38.1 202.7 1005.2 2037.3

P&A Batch-4 19.6 41.6 199.3 994.2 2019.7

P&A Batch-5 20.5 38.2 198.3 1000.8 1996.6

N 5 5 5 5 5

Mean 20 39.8 201.8 1001.2 1997.5

SD(±) 0.4 1.6 3.7 6.2 34.4

CV% 2 4 1.8 0.6 1.7

%Nominal 100.0 99.5 100.9 100.1 99.9

Analytical run

ID

Nominal Concentration (ng/ml)

CS6 CS7 CS8 CS9 CS10

4000.00

pg/ml

8000.00

pg/ml

12000.00

pg/ml

16000.00

pg/ml

20000.00

pg/ml

P&A Batch-1 4086 8124.8 11826.7 15539.5 19778.9

P&A Batch-2 4041.4 8119.9 11873.8 15672.1 19981.2

P&A Batch-3 4008.5 7955.2 11955 15737.6 20228.6

P&A Batch-4 4005.1 8042.7 11843.5 16065 19624.9

P&A Batch-5 4024.8 8068.4 11851.2 16469.8 19980.9

N 5 5 5 5 4

Mean 4033.2 8062.2 11870 15896.8 19918.9

SD(±) 32.9 69.1 50.4 374.2 229

CV% 0.8 0.9 0.4 2.4 1.1

%Nominal 100.8 100.8 98.9 99.4 99.6

Acceptance criteria

1. Mean % Nominal (100±15%) except lowest calibration standard.

2. Mean % Nominal (100±20%) for lowest calibration standard (CS1).

3. %CV ≤ 15% except lowest calibration standard (CS1) for which it is ≤20%.


155

Extraction Recovery

The percentage recovery of Rifaximin was determined by comparing the mean

peak area of Rifaximin in extracted LQC, MQC, HQC samples with freshly prepared

unextracted LQC, MQC, HQC samples respectively.

The mean % recovery for LQC, MQC, HQC samples of Rifaximin were

86.30%, 87.58% and 88.72% respectively.

The mean recovery of Rifaximin across QC levels is 87.53%.

The mean recovery of % CV recovery of Rifaximin across QC levels is 4.5%.

For the internal standard, mean peak area of 18 extracted samples was

compared to the mean peak area of 18 unextracted IS solution. The mean %

recovery is 93.34%.

The %CV recovery of IS Rifaximin D6 for extracted is 4.6%.

Results are presented in 5.10.


156

Table 5.10: Recovery of analyte (Rifaximin) and IS (Rifaximin–D6) from human

plasma

Standard

Extracted peak

response

Unextracted peak

response

Drug IS Drug IS

Low QC: 60.00 ng/ml

6847 262864 8100 283969

6536 255326 7470 270810

6637 251134 8120 276140

6950 266426 8185 304577

7243 270531 8320 301416

7431 291083 8064 296283

N 6 6 6 6

Mean 8043.2

SD (±) 294.90

%CV 3.7

Medium QC: 6000.00 ng/ml

676325 259753 783059 280026

681889 266219 743270 273798

703849 269736 760476 274662

692229 264352 829485 296621

716578 269336 854206 303456

735814 282716 833567 297277

N 6 6 6 6

Mean 800677.2

SD(±) 44719.31

%CV 5.6

High QC: 14000.00 ng/ml

1700730 275722 1808893 279569

1522451 248966 1746052 269729

1582082 263536 1828022 286119

1711926 283786 1915258 297319

1602680 263905 1929544 295458

1754556 293154 1905470 296383

N 6 6 6 6

Mean 1855539.8

SD(±) 72741.52

%CV 3.9

Drug IS

Mean recovery of across QC levels 87.53 93.34

Mean SD(±) of across QC levels 3.966 4.328

The Mean % CV across QC levels 4.5 4.6


1. The coefficient of variation for mean recovery across LQC, MQC and HQC

shall not exceed 25%.

2. The coefficient of variation for mean recovery of IS shall not exceed 25%.


157

Matrix Effect

Samples were prepared at MQC level in triplicate in each of 6 different lots of

human plasma. A calibration curve and 6 replicates of MQC samples in triplicate for

each matrix were freshly prepared and analyzed in single run.

Percentage bias was calculated for each matrix.

No significant matrix effect found in different sources of human plasma tested

for Rifaximin, Rifaximin D6.

Results are presented in table 5.11 and 5.12.


158

Table 5.11 Assessment of Matrix Effect on determination of Rifaximin levels in

human plasma

Identification of

matrix

Drug response

in Matrix at

MQC Level

Drug response in

No Matrix at

MQC Level

Matrix factor

AP/2451/09/10 294100 308805 0.779

AP/2451/09/10 308500 323925 0.8197

AP/2451/09/10 290200 304710 0.8039

AP/2452/09/10 289000 303450 0.7888

AP/2452/09/10 269000 282450 0.7717

AP/2452/09/10 292300 306915 0.7952

AP/2453/09/10 308200 323610 0.8046

AP/2453/09/10 302600 317730 0.8601

AP/2453/09/10 269900 283395 0.7826

AP/2454/09/10 301800 316890 0.8737

AP/2454/09/10 299600 314580 0.8541

AP/2454/09/10 322600 338730 0.8417

AP/2455/09/10 303700 318885 0.8109

AP/2455/09/10 319900 335895 0.8373

AP/2455/09/10 312100 327705 0.8457 AP/2456/09/10 330300 346815 0.8756

AP/2456/09/10 307300 322665 0.7842

AP/2456/09/10 321200 337260 0.8518 N 18 18 18

Grand Mean 0.8211 SD(±) 0.0343

CV (%) 4.18


1. Mean % Nominal 100±15% of nominal value.

2. %CV ≤ 15%.


159

Table 5.12 Assessment of Matrix Effect on determination of IS

(Rifaximin D6) levels in human plasma at MQC Level

Identification of matrix Internal

standard

response in

matrix at MQC

level

Internal

standard

response in No

matrix

at MQC level

Matrix factor

AP/2451/09/10 377528 415281 0.909091

AP/2451/09/10 376377 414015 0.909091

AP/2451/09/10 361012 397113 0.909091

AP/2452/09/10 366363 402999 0.909091

AP/2452/09/10 348576 383434 0.909091

AP/2452/09/10 367568 404325 0.909091

AP/2453/09/10 383069 421376 0.909091

AP/2453/09/10 351801 386981 0.909091

AP/2453/09/10 344879 379367 0.909091

AP/2454/09/10 345431 379974 0.909091

AP/2454/09/10 350778 385856 0.909091

AP/2454/09/10 383293 421622 0.909091

AP/2455/09/10 374532 411985 0.909091

AP/2455/09/10 382056 420262 0.909091

AP/2455/09/10 369063 405969 0.909091

AP/2456/09/10 377212 414933 0.909091

AP/2456/09/10 391857 431043 0.909091

AP/2456/09/10 377099 414809 0.909091 N 18 18 18

Grand Mean 0.909090909 SD(±) 8.07806E-17 %CV 8.88586E-15

Acceptance criteria: %CV<25%.


160

Dilution Integrity

Dilution integrity experiment was carried out at six replicate of two times

diluted (1 in 2 dilution) and four times diluted of approx 1.5 × ULOQ (1 in 4 dilution)

samples were prepared and concentrations were calculated including the dilution

factor against the freshly prepared calibration curve.

The % accuracy of Rifaximin nominal concentrations ranged between 96.17%

and 97.67% for 1 in 2 dilutions and 1 in 4 dilutions respectively.

The % CV is 1.46% to 1.48%.

Results are presented in tables 5.13.

Table 5.13 Assessment of Dilution integrity for Rifaximin

at DQC Conc (pg/ml)

DQC

Dilution factor: ½

Nominal conc: 30000.00 pg/ml

DQC

Dilution factor: ¼

Nominal conc: 30000.00 pg/ml

Conc. Found % Nominal Conc. Found %Nominal

28900 96.33 29200 97.33 28500 95.00 28800 96.00 28300 94.33 28800 96.00 29100 97.00 29600 98.67 28800 96.00 29700 99.00 29500 98.33 29700 99.00

N 6

6

Mean

%Nominal 96.17 97.67

SD (±) 1.43 1.43

CV (%) 1.48 1.46


1. % CV ≤ 15%.

2. Mean % Nominal (100±15%).


161

Whole Batch Reinjection Reproducibility

To evaluate the whole batch reinjection reproducibility experiment, samples of

P & A batch-2 were kept at auto sampler temperature for approx 26 hrs after the

initial analysis and were re-injected again after approx 26 hrs. Concentrations were

calculated to determine precision and accuracy after reinjection.

The Accuracy of Rifaximin QC samples in reinjection was between 86.12%

and 99.39%.

The Precision (%CV) of Rifaximin QC samples in reinjection was between

1.10 % and 2.75%.

Rifaximin was found to be stable at autosampler temperature post extraction

(in reconstitution solution) for approx 26 hrs and reproducible after

reinjection.

Results are presented in tables 5.14.


162

Table 5.14 Assessment of Whole Batch Reinjection Reproducibility during

estimation of Rifaximin in human plasma

Analytical

Run ID

Low QC 60.00 pg/ml High QC 14000.0 pg/ml

Comp sample Reinjection

sample Comp sample

Reinjection

sample

66.8 58.4 13500 13100 66.7 58 13300 13400 71.2 62 13600 13400 67.3 58.5 13400 13200 70.2 59.4 13500 13500 68.7 57.5 13200 13400

N 6 6 6 6

Mean 68.48 58.97 13416.67 13333.33 SD(±) 1.89 1.61 147.20 150.55 %CV 2.75 2.74 1.10 1.13

%NOM 86.12 99.39


1. % CV≤ 15% Except LLOQ for which it is ≤ 20%.

2. Mean % Nominal (100±15% and for LLOQ 100±20%).

3. 67% 0f the re-injected QCs at each level shall be within ±20% of their

previous concentration.

Ruggedness-Different Analyst

To evaluate ruggedness experiment with different analysts, one P&A batch

(P&A-3) was processed by different analyst. The run consisted of a calibration curve

standards and 6 replicates of each LLOQ, LQC, MQC, HQC samples.

The Accuracy of Rifaximin QC samples within the range of 89.75% to

101.04%.

The Precision of Rifaximin QC samples within the range of 0.61% to 5.20%.


163

These results indicated that the method is rugged and reproducible by different

analyst.

Results are presented in tables 5.15

Table 5.15 Ruggedness of the method for estimation of Rifaximin Plasma levels

in human plasma with different analyst.

Analytical

Run ID

LLOQ

20.00 ng/ml

Low QC

60.00 ng/ml

Mid QC

700.00 ng/ml

High QC

1400.00 ng/ml

Analyst

ID A

Analyst

ID B

Analyst

ID A

Analyst

ID B

Analyst

ID A

Analyst

ID B

Analyst

ID A

Analyst

ID B

P&A Batch

3

21.20 19.10 61.90 60.40 6135.20 6094.50 13380.10 13384.50 22.80 18.50 60.20 59.00 6011.10 6072.80 13334.40 13331.20 21.10 19.40 65.10 60.60 6181.90 6049.30 13323.50 13370.10 21.00 19.40 61.80 61.00 6164.10 6177.90 13489.70 13754.60 22.00 20.80 62.70 64.80 6209.60 6064.50 13522.70 13532.10 21.70 19.30 63.30 67.50 6122.70 6055.90 13378.50 13894.80

N 6 6 6 6 6 6 6 6

Mean 21.63 19.42 62.50 62.22 6137.43 6085.82 13404.82 13544.55

SD (±) 0.69 0.76 1.65 3.24 69.40 47.76 82.43 231.74

CV (%) 3.18 3.90 2.64 5.20 1.13 0.78 0.61 1.71

%Accuracy 89.75 99.55 99.16 101.04



2. Mean % Nominal (100±15% & for LLOQ 100±20%).


164

Ruggedness-Different Column

To evaluate ruggedness experiment with different column, samples of P&A

batch-5 were reinjected on different columns with same and specifications,

Concentrations were calculated to determine precision and accuracy.

The Accuracy of Rifaximin QC samples within the range of 94.56% to

102.36%.

The Precision of Rifaximin QC samples within the range of 0.78% to

8.01%.

These results indicated that the method is rugged and reproducible by different

analyst.

Results are presented in tables 5.16

Table 5.16 Ruggedness of the method for estimation of Rifaximin Plasma levels

in human plasma with different Analytical column

Analytical

Run ID

LLOQ

20.00 ng/ml

Low QC

60.00 ng/ml

Mid QC

700.00 ng/ml

High QC

1400.00 ng/ml

Column

ID

LC/171

Column

ID

LC/182

Column

ID

LC/171

Column

ID

LC/182

Column

ID

LC/171

Column

ID

LC/182

Column

ID LC/171

Column

ID LC/182

P&A Batch

5

18.80 19.10 57.90 60.40 5819.50 6094.50 12506.90 13384.50 19.20 18.50 58.10 59.00 5960.90 6072.80 12704.80 13331.20 19.50 19.40 62.30 60.60 6001.30 6049.30 12967.20 13370.10 20.80 19.40 61.10 61.00 6113.20 6177.90 13532.30 13754.60 22.80 20.80 62.70 64.80 6177.30 6064.50 13758.10 13532.10 22.10 19.30 62.60 67.50 6496.10 6055.90 13942.00 13894.80

N 6 6 6 6 6 6 6 6

Mean 20.53 19.42 60.78 62.22 6094.72 6085.82 13235.22 13544.55 SD(±) 1.64 0.76 2.23 3.24 232.66 47.76 590.73 231.74

CV (%) 8.01 3.90 3.67 5.20 3.82 0.78 4.46 1.71 %Accuracy 94.56 102.36 99.85 102.34



2. Mean % Nominal (100±15% & for LLOQ 100±20%).


165

Bench Top Stability (at room temp for 24.5 hrs)

Spiked LQC and HQC samples were retrieved from deep freezer and were kept at

room temperature for 24.5 hrs and were processed and analyzed along with freshly prepared

calibration standards, comparison LQC and HQC samples. Concentrations were calculated to

determine mean % change during stability period.

The mean Accuracy for LQC & HQC samples of Rifaximin from comparison

samples were 94.52% and 101.81% respectively.

The plasma samples of Rifaximin were found to be stable for approximately

24.5 hrs min at room temperature.

Results are present in table 5.17.

Table 5.17 Assessment of stability of Analyte (Rifaximin) in Biological matrix at

Room temperature


Comparison

samples

(0.00 hr)

Stability samples

(24.5 hrs)

Comparison

samples

(0.00 hr)

Stability samples

(24.5 hrs)

Conc.

found

%

nominal

Conc.

found

%

nominal

Conc.

found

%

nominal

Conc.

found

%

nominal

57.9 96.4 59.2 98.67 12500 89.3 13000 92.86 58.1 96.9 56.9 94.83 12700 90.7 13100 93.57 62.3 104 61.6 102.67 13000 92.6 13000 92.86 61.1 102 61.6 102.67 13500 96.7 13700 97.86 62.7 105 62.8 104.67 13800 98.3 13900 99.29 62.6 104 64.4 107.33 13900 99.6 14300 102.14

N 6

6

6

6

Mean 60.78 61.08 13233.33 13500.00 SD(±) 2.23 2.67 585.38 547.72

CV (%) 3.67 4.36 4.42 4.06 %Accuracy 101.31 101.81 94.52 96.43


1. % Ratio (stability/comparison) should be within 85-115 %.

2. %CV ≤ 15%.

3. Mean % Nominal (100±15%).


166

Freeze and Thaw Stability (after 3rd

cycle at -30°C)

Samples were prepared at LQC and HQC levels, aliquoted and frozen at -

30±5°C six samples from each concentration were subjected to three freeze and thaw

cycles (stability samples). These samples were processed and analyzed along with

freshly prepared calibration standards, LQC and HQC samples (comparison samples).

Concentrations were calculated to determine mean % change after 3 cycles.

The mean Accuracy for LQC & HQC samples of Rifaximin from comparison

samples were 98.97% and 104.01% respectively.

The plasma samples of Rifaximin were found to be stable after 3 cycles at -

30±5°C.


Table 5.18 Assessment of Freeze-Thaw stability of Analyte (Rifaximin)

at -30±5°C


Comparison

samples

Stability sample

at 4th

cycle

Comparison

samples

Stability sample at

4th

cycle

Conc.

found

%

nominal

Conc.

found

%

nominal

Conc.

found

%

nominal

Conc.

found

%

nominal

57.90 96.40 56.30 112.60 12500.00 89.30 12800.00 91.43 58.10 96.90 57.60 115.20 12700.00 90.70 13100.00 93.57 62.30 104.00 59.00 118.00 13000.00 92.60 13100.00 93.57 61.10 102.00 60.30 120.60 13500.00 96.70 13800.00 98.57 62.70 105.00 61.30 122.60 13800.00 98.30 14000.00 100.00 62.60 104.00 63.30 126.60 13900.00 99.60 14000.00 100.00

N 6

6

6

6

Mean 60.78 59.63 13233.33 13466.67 SD(±) 2.23 2.54 585.38 527.89

CV (%) 3.67 4.26 4.42 3.92 %Accuracy

101.38 119.21 94.53 96.19

Acceptance criteria

Same as 5.17


167

Autosampler stability at 2-8°C in autosampler

LQC and HQC samples were prepared and processed. These processed

samples were analyzed and kept in auto sampler for 59 hrs at 2-8°C and analyzed

along with freshly prepared calibration standard samples. Concentrations were

calculated to determine mean % change during stability period.

The mean Accuracy change for LQC & HQC samples of Rifaximin from

comparison samples were 102.50% and 105.69% respectively.

Rifaximin samples were stable for 59 hrs at 2-8°C in autosampler.

Results are present in table 5.19

Table 5.19 Assessment of autosampler stability of Analyte (Rifaximin)

at 2-8°C


Comparison

samples (0.0 hr)

Stability samples

(59 hr)

Comparison

samples (0.0 hr)

Stability samples

(59 hr)

Conc.

found

%

nominal

Conc.

found

%

nominal

Conc.

found

%

nominal

Conc.

found

%

nominal

57.90 96.40 57.8 96.33 12500.00 89.30 13200 94.29 58.10 96.90 61.6 102.67 12700.00 90.70 13100 93.57 62.30 104.00 58.3 97.17 13000.00 92.60 12900 92.14 61.10 102.00 63.5 105.83 13500.00 96.70 13800 98.57 62.70 105.00 64.5 107.50 13800.00 98.30 14100 100.71 62.60 104.00 64.4 107.33 13900.00 99.60 13900 99.29

N 6

6

6

6

Mean 60.78 61.68 13233.33 13500.00

SD(±) 2.23 3.00 585.38 493.96

CV (%) 3.67 4.87 4.42 3.66

%Accuracy 101.38

102.81

94.53

96.43

Acceptance criteria: Same as 5.17


168

Long-term stability (at -30°C temp for 55 days)

Spiked LQC and HQC samples were retrieved from deep freezer after 55 days

and were processed and analyzed along with freshly prepared calibration standards,

comparison LQC and HQC samples. Concentrations were calculated to determine

mean % change during stability period.

The mean Accuracy for LQC and HQC samples of Rifaximin from

comparison samples were 98.89% and 96.31% respectively.

The plasma samples of Rifaximin were found to be stable for approximately

55 days at -30°C temp.


Table 5.20 Assessment of long term plasma stability of analyte

(Rifaximin) at -30°C.

Low QC 60.00 pg/ml High QC 14000.0 pg/ml Comparison

samples (0.0 hr)

Stability samples

(55 days)

Comparison samples

(0.0 hr)

Stability samples (55

days)

Conc.

found

%

nominal

Conc.

found

%

nominal

Conc.

found

%

nominal

Conc.

found

%

nominal

57.90 96.40 56 93.33 12500.00 89.30 12900 92.14 58.10 96.90 57.4 95.67 12700.00 90.70 13100 93.57 62.30 104.00 58.3 97.17 13000.00 92.60 12900 92.14 61.10 102.00 59.2 98.67 13500.00 96.70 13900 99.29 62.70 105.00 62.4 104.00 13800.00 98.30 13800 98.57 62.60 104.00 62.7 104.50 13900.00 99.60 14300 102.14

N 6

6

6

6

Mean 60.78 59.33 13233.33 13483.33 SD(±) 2.23 2.71 585.38 594.70 CV (%) 3.67 4.56 4.42 4.41

%Accuracy 101.38

98.89

94.53

96.31

Acceptance criteria: Same as 5.17


169

Short Term Stock Solution Stability of Rifaximin, and Rifaximin D6 at Room

Temperature

Stock solution stability was determined by comparing the peak areas of freshly

prepared stock solutions (comparison samples) with stability stock solutions. Main

Stock solutions of Rifaximin and Rifaximin-D6 were freshly prepared and aliquots of

stocks were kept at room temperature for 9.5 hr (stability samples). Aqueous

equivalent highest calibration standard of Rifaximin and solution of Rifaximin D6

were prepared from the stability samples and analyzed. Areas of stability samples and

freshly prepared samples were compared to determine mean % change during stability

period.

The % CV for of Rifaximin stock solution from comparison samples was

13.00% and % Ratio (stability/comparison) was 102.65

The % CV for of Rifaximin D6 stock solution from comparison samples was

7.00% and % Ratio (stability/comparison) was 86.23

The % CV for Rifaximin D6 working solution (Internal standard spiking

solution) from comparison samples was 8.4% and % Ratio (stability/

comparison) was 96.71

Rifaximin, Rifaximin D6 stock solutions and Rifaximin D6 spiking solutions

were found to be stable at room temperature for 9.5 hr.

Results are present in table 5.21 and 5.22.


170

Table 5.21 Assessment of Short term stock solution stability of Analyte

(Rifaximin) and Internal standard (Rifaximin D6) at Room temperature

Analyte Internal standard Comparison

Standard stock

solution response (0.0

hr)

Stability stock

solution response

(9.5 hr)

Comparison stock

solution response

(0.0 hr)

Stability

Standard stock

Response

(9.5 hr)

474501 494198 149258 114253

403394 424176 144813 115052

364237 395234 119758 127061

352525 369730 140339 106238

401340 391055 137944 127437

358583 342676 133735 122088

N 6 6 6 6

Mean 392430.00 402844.83 137641.17 118688.17

SD (±) 45756.136 52372.178 10284.180 8319.908

CV (%) 11.7 13.0 7.5 7.0

%

Ratio 102.65 86.23


1. % change should be ± 15 %


171

Table 5.22 Assessment of short term solution stability of internal standard

spiking solution (Rifaximin D6) at room temperature

Comparison solution (Internal

standard Spiking solution)

Response (0.0 hr)

Stability solution (Internal

standard spiking solution)

Response (9.5 hr)

128767 129387

109739 123765

128790 106997

112539 124138

122194 110336

123926 107481

N 6 6

Mean 120992.50 117017.33

SD (±) 8116.598 9851.218

CV (%) 6.7 8.4

% Ratio 96.71


1. % change should be ± 5%

Method validation Conclusion

As all the values obtained were within the Acceptance criteria. The method

stands validated and is suitable for estimation of plasma concentrations of Rifaximin

in a single analytical run. The rugged, efficient Liquid-liquid extraction method

provides exceptional sample clean up and constant recoveries using 400µl of plasma.

The high extraction efficiency, low limit of quantification, and wide linear dynamic

range make this a suitable method for use in clinical samples from Pharmacokinetic

studies following oral administration of Rifaximin fixed dose (200/200 mg) tablets in

healthy human subjects.


172

5.5 Applications

The analytical method described above was used to determine Rifaximin

concentrations in plasma following oral administration of healthy human volunteers.

Each volunteer obtained written informed consent before participating in this study.

These volunteers were contracted in APL Research centre, India, and 200 mg dose

(one 200mg Tablet) was administered in 17 healthy volunteers by oral administration

with 240 mL of drinking water. The reference product xifaxan Tablets (Salix

Pharmaceuticals, USA) 200 mg and test product Rifaximin tablet (Test Tablet) 200

mg was used. Study protocol was approved by Indian Ethical Committee (IEC) as

per Indian Council of Medical Research (ICMR). Blood samples were collected as

pre-dose(0) h 5 min prior to dosing followed by further samples at 0.25, 0.5, 0.75, 1,

1.25, 1.5, 1.75, 2, 2.5, 3, 4, 6, 9, 12, 16, 20, and 24.0 h. After dosing, 7 ml blood was

collected each time in vaccutainers containing K2EDTA. A total of 36 (18 time points

for test and 18 time points for reference) time points were collected by using

centrifugation 3200 rpm, 10°C, 10 min, and kept frozen at -30 °C until sample

analysis. Test and reference were administered to the same human volunteers under

fasting conditions separately with proper washing periods as per approved protocol.

The Mean Plasma concentration data for 17 volunteers is represented in Table

5.23 with respective concentration-time curve is shown in Figure 5.15


173

Figure 5.15 Mean plasma Concentration – Time curve for Rifaximin

Mean plasma concentration of Rifaximine

0

200

400

600

800

1000

1200

1400

0 5 10 15 20 25 30

Time (hr)

Rif

axim

ine(

pg

/mL

)

Test

Ref


174

Table 5.23. Rifaximin Mean Concentration (pg/ml) data of the subject samples obtained

from the HPLC-MS/MS

Time in hours Mean Plasma Concentration data

Test Reference

0 0.00 0.00

0.25 132.02 84.86

0.5 788.82 427.46

0.75 1063.49 650.08

1 1236.54 855.46

1.25 1277.59 940.88

1.5 1185.74 910.38

1.75 1148.23 850.87

2 1055.61 775.46

2.5 930.79 749.64

3 798.47 620.21

4 608.42 534.75

6 501.48 421.35

9 336.55 273.99

12 247.64 210.09

16 170.82 152.19

20 138.77 117.18

24 101.74 98.69


175

5.6 Pharmacokinetic Studies

Pharmacokinetic parameters from the human plasma concentration samples

were calculated by a non compartmental statistics model using WinNon-Lin5.0

software (Pharsight, USA). Blood samples were taken for a period of 3 to 5 times of

the terminal elimination half-life (t1/2) and it was considered as area under the

concentration time curve (AUC) ratio higher than 80% as per FDA guidelines8-10

.

Plasma Rifaximin concentration-time profiles were visually inspected Cmax and Tmax

values were determined. The AUC0–t was obtained by trapezoidal method. AUC0-∞

was calculated up to the last measureable concentration and extrapolations were

obtained using the last measureable concentration and the terminal elimination rate

constant (Ke). The terminal elimination rate constant (Ke), was estimated from the

slope of the terminal exponential phase of the plasma of Rifaximin concentration–

time curve by means of the linear regression method. The terminal elimination half-

life, t1/2, was then calculated as 0.693/Ke. Regarding AUC0–t, AUC0-∞ and Cmax

bioequivalence was assessed by means of analysis of variance (ANOVA) and

calculating the standard 90% confidence intervals (90% CIs) of the ratios

test/reference (logarithmically transformed data). The bioequivalence was considered

when the ratio of averages of log-transformed data was within 80 to 125% for AUC0–t,

AUC0-∞ and Cmax. Pharmacokinetic data is shown in Table 5.24 and Table 5.25.


176

Table 5.24 Rifaximin Pharmacokinetic data

Rifaximin Pharmacokinetic data

Pharmacokinetic

Parameter

Test Reference

Mean±SD Mean±SD

Cmax 1277.59±453.42 940.88 ± 323.60

AUC 0-t 8652.74 ± 346.74 6998.43 ± 305.93

AUC 0-inf 9959.52 8250.37

tmax 1.25 1.25

T 1/2 8.90 8.79

Table 5.25. Rifaximin Pharmacokinetic data

Pharmacokinetic

Parameter

Cmax AUC 0-t AUC 0-inf

Test/Reference 135.78 123.64 120.72

Pharmacokinetic Studies Conclusion

The present study provides firm evidence to support that the in house

Rifaximin 200 mg was not bioequivalent with xifaxan Tablets (Salix Pharmaceuticals,

USA) 200 mg tablet under fasting conditions.

In vivo data was predicted by using Liquid Liquid Extraction procedure and

concentrations were found through Liquid Chromatography Tandem Mass

Spectroscopy detection. The Pharmacokinetic parameters assessed were AUC0-t,

AUC0-, Cmax, Tmax, t1/2. The bioequivalence criteria are based on the 90% confidence

intervals whose acceptance range is in between 80%-125%.

The results obtained for Rifaximin was not within the acceptance range.

Therefore, it can be concluded that the two Rifaximin formulations (reference and

test) analyzed were not bioequivalent in terms of rate and extent of absorption. Based

on the pharmacokinetic parameters it is decided that the developed test formulation of

Rifaximin need to be redeveloped in terms of formulation aspect.

5.1 introduction - shodhgangashodhganga.inflibnet.ac.in/bitstream/10603/11199/9/chapter - 5.pdf ·...

Documents