Devanshu Final Ppt.1

8/3/2019 Devanshu Final Ppt.1

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BIOANALYTICAL METHOD DEVELOPMENT AND

VALIDATION FOR THE ESTIMATION OF MOXIFLOXACIN IN

HUMAN PLASMA USING LIQUID CHROMATOGRAPHY-TANDEM MASS SPECTROMETRY(LC-MS/MS)

Presented By:Devanshu Sharma

M.Pharm 2nd

yr(Pharmaceutics)MMU, Mullana.

1

Guide:

Dr. Anroop B Nair(Professor)

M.M College of Pharmacy

Co-Guide:

Dr. Sanjay Gurule(Group Leader)

Ranbaxy Research Laboratories


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1.Introduction

Bioanalysis is sub disciplined of analytical chemistry covering the

quantitative measurement of xenobiotics (drugs and their metabolites) in

biological system.

Bioanalytical method -set of all procedures involved in the collection,

processing, storing and analysis of biological matrix for an analyte.

LC-MS/MS is currently considered as a important analytical tool forquantitative analysis of drug(s) and metabolite(s) in biological fluids.

Method development involves evaluation and optimization of the various

stages of sample preparation, chromatographical separation, detection and

quantification.

Method validation includes all of the procedures required to demonstratethat a particular method is reliable for the intended application.

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2.Objective

The objective of this study is to establish a simple, accurate, rapid and

sensitive method using LC-MS/MS for the quantification of Moxifloxacin

in human plasma as per USFDA guidelines.

Supportive Objectives:

To select sample processing and extraction process for Moxifloxacin

extraction from human plasma. To select good column which will solve the problem of low response.

To select the appropriate mobile phase, reconstitution solution, rinsing

solution which will solve the problems of tailing of peak, carry over effect

respectively.

The developed and validated bio analytical method is used in performingbioequivalence study between the reference drug and generic drug

formulation.

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4

PLAN OF WORK

Identification of Drug.

Bio-analytical method development and validation.

Application of developed bio-analytical method to pharmacokinetic studies.


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Method Development

Method development is the setting up of an analytical procedure that will be appropriate for the

analysis of a particular sample.

Analytical strategy

The choice of method involves several considerations:

Which concentrations of the compound will be present in the samples?

What is the sample matrix?

What is the cost of the analysis?

Sample collection

Biological samples used in assays for the determination of drugs are often venous blood withdrawn

from the arm. The venous blood can be withdrawn into tubes with an anticoagulant, e.g. EDTA and

heparin. Both blood and plasma can be used for analysis.

Sample preparation Protein precipitation

Liquid-liquid extraction

Solid-phase extraction

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General method development steps include:-

a) Scanning

b) Optimization of LC-MS/MS parameter

c) Selection and optimization of mobile phase

d) Selection of column

e) Selection and optimization of sample

preparation method

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4. Drug profile

Fig. Molecular structure of Moxifloxacin

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Method Development

Scanning and Optimization on instrument

Stock of moxifloxacin and moxifloxacin d-4 (1mg/ml) and its two metabolites i.e moxifloxacin

acyl B-D glucuronide and moxifloxacin- n sulphate were weighed and dissolved in methanol

then for scanning of drug make dilution of drug 500ng/ml in (50:50) methanol and water.

Scanning of Moxifloxacin and Moxifloxacin d-4 was performed in +ve ion mode, m/z were

Moxifloxacin 402.2/358.3 and Moxifloxacin d-4 406.2/362.2 . Scanning and Optimization for

Moxifloxacin and Moxifloxacin d-4 was done in methanol: water (50:50). Each ions were

scanned at different m/z ratios .

Selection of column

Various columns selected for the analysis includes Discovery-C18 ,Ascentis-C18,Chromolith

performance were tried but less response and saturation in calibration curve was observed. But

in chromolith performance (100x4.6mm) there was good resolution as compared to other

columns used, desired retention time and symmetric peak shape was obtained .

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Table : m/z ratio of ions of Moxifloxacin and Moxifloxacin d-4

m/z ratios Q1 scan (Parent) Q3 scan (Product)

Moxifloxacin 402.2 358.3

Moxifloxacind-4Moxifloxacin acyl-b-d glucuronideMoxifloxacin N-sulphate

406.2578.3482.2

362.2384.3402.3


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Detection

Mass spectrometer was used as a detector for the detection of the parent ion & product ion

according to their m/z values

MS detector consists of three main parts: the interface where the ions are generated, the mass

analyzer (separation) and the electron multiplier (detector)

The most popular ionization techniques are: electro spray ionization (ESI), atmospheric pressurechemical ionization (APCI)

The most common mass analyzers are: Triple quadrupole, ion-trap.

Selection of mobile phase

Various buffers (such as Ammonium Formate 1, 2,5mM) with 0.2% formic acid were made in

combination with Methanol and Acetonitrile in varying ratios (20:80, 30:70, 40:60) as a mobile

phase but there was a good chromatogram obtained in 5mM Ammonium formate(0.2% formicacid)-60:40-(Methanol).In Ammonium acetate there was a very bad peak shape with tailing

effect. Column chromloith performance was used which yielded good results. Then one P.A

batch was processed.

Selection of Internal standard

Compound with similarity in structure and physicochemical properties was tried as internal

standard which would give sufficient retention and stable response were obtained withmoxifloxacin d-4.

Selection of sample processing method

Solid Phase Extraction: Sample processing carried out by Solid Phase Extraction withBond Elute Plexa cartridges, using HPLC grade water (1ml) for washing twice and methanol

(1ml ) for elution(single) and centrifuge at 1500 rpm for 1 minute.

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6.Sample Extraction Procedure

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PREPARATION OF REAGENTS :-

Ammonium Formate buffer:Transferred 0.315g of ammonium formate into reagent bottleand added 1000ml of HPLC grade water and mixed well by sonicating in an ultrasonic bath to

get 5.0 0.1mM ammonium formate buffer solution. Adjust the pH to 2.600.1 with formic acid

Mobile phase:Transferred 600ml of Ammonium formate buffer into a 1000-mL reagentbottle, and added 400 mL of HPLC grade methanol.

Diluent-1:Transferred 500 mL of HPLC grade methanol into a 1000-mL reagent bottle andadded 500 mL of HPLC grade water.

Rinsing solution:500ml methanol+500 ml of HPLC grade water.

Preparation of internal standard solution (IS dilution) Weighed accurately Moxifloxacin d-4 internal standard and transferred into a volumetric flask.Dissolved in sufficient volume of methanol and made up the volume with the same to produce

1mg/mL of Moxifloxacin d-4. The stock solution was further diluted with methanol and water

(50:50) to get the final concentration of 2000ng/ml.

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Preparation of Moxifloxacind-4 stock solution

Amt. Weighed (mg) 1

Volume made up to (ml) 1

Potency (%) 100

Mol wt of compound 441.92

Mol wt of Free compound N/AP

Calculated stock conc.(ng/ml)

1000000

Table : Preparation of Moxifloxacind-4 stock dilution

Stock Conc. Stock Aliquot Total Volume IS Dilution

(ng/mL) (mL) (mL) Conc. (ng/mL)

1000000 0.040 20 2000


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7. Optimized Chromatographic condition

Parameter Details

Column Chromolith Performance, RP-18,1004.6mm

Mobile Phase Methanol: Buffer-1** (40:60, v/v)

Reconstitution solution Methanol: Solution-1*(40:60, v/v)

Rinsing solution Methanol: HPLC grade water (50:50, v/v)

Flow Rate 1.2ml/minute

Split Ratio Approximately 0.400 ml mobile phase shouldgo to the ion source per minute

Sample cooler temperature 5C 1.0C

Column oven temperature 45C 1.0C

Injection Volume 10l

Retention time Moxifloxacin:1.40 to 2.40 minutesMoxifloxacin-d4:1.4 to 2.40 minutes

Ion source(polarity) Turbo-ion spray in positive ion mode

* Solution-1:Methanol: 500 ml hplc grade water with triethyl amine(.pH 10.5)

**Buffer-1:Methanol: Ammonium Formate (5mM,pH 2.60) 12


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8.Instrument specifications

Parameter Specification

Ion spray voltage 5500V

Temperature 450C

Curtain gas 8

Nebulizer gas 10Scanning mode Positive mode

Detection:

Moxifloxacin m/z 402.2 Q1(Mass) and 358.30 Q3

(Mass)

Moxifloxacin-d4 m/z 406.2 Q1(Mass) and 362.20 Q3

(Mass)13


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Stat file information for API 3000

Detection: Moxifloxacin m/z 402.2 / 358.3

Moxifloxacin d-4 m/z 406.2 / 362.2

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Source Parameters of Moxifloxacin andMoxifloxacin d-4

Parameters Value

NC (Nebulizer

current)

10V

TEM(Temperature)

500C

CUR (Curtaingas)

08#

CAD 10#

# - these are typically values based on setting and scale defined insoftware application to control gas parameters

Compound Parameters of Moxifloxacin andMoxifloxacin d-4

Parameters Moxifloxacin Moxifloxacin d-4

DP (Declusteringpotential)

40V 40V

EP (Entrancepotential)

10V 10V

CE (Collisionenergy)

29V 29V

CXP (Collision cellexit potential)

25V 25V


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Preparation of Calibration Curve and quality controls Standard Preparation of standard stock solution of Moxifloxacin for CC:

Weighed accurately 1mg of moxifloxacin working standards and transferred into a 1mL

volumetric flask. Dissolved in sufficient volume of methanol and made up to the volume with

the same to produce 1mg/mL stock solution of moxifloxacin. Stored in refrigerator below 8 C.The stock solution was diluted to suitable concentrations using Diluent-1 for dilution for spiking

in plasma to obtain calibration curve (CC) standards.

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Table : Preparation of moxifloxacin stocksolution

Amt. Weighed (mg) 1


Potency (%) 100


Free Mol wt of compound N/AP

Calculated stock conc.(ng/mL)

1000000

(

Table : Preparation of standard stock dilutions ofmoxifloxacin for CC

Stock Conc.(ng/mL)

Stock Aliquot(mL)

Volume Madeupto (mL)

Stock DilutionConc. (ng/mL)

Stock Dilution ID

1000000 1.5 5 300000AQ-H

300000 4 5 240000AQ-G

240000 2.5 5 120000AQ-F

120000 2.5 5 60000AQ-E

60000 2 5 24000AQ-D

24000 2 5 9600AQ-C

9600 2 5 3840AQ-B

3840 2 5 1536AQ-A


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Spiking of Dilutions in plasma for calibration curve standards

Transfer 0.2ml of each of above stock dilution of moxifloxacin into 10ml volumetric flask and

made up volume with plasma to achieve concentration ranging from 30.7 to 6000 ng/ml

Preparation of standard stock solution of Moxifloxacin for QC Weighed accurately 1 mg of Moxifloxacin working standard and transferred into a 1mL

volumetric flask. Dissolved in sufficient volume of methanol and made up to the volume with

the same to produce 1 mg/mL stock solution of Moxifloxacin. Stored in refrigerator below 10

C . The stock solution was diluted to suitable concentrations using diluent-1 for dilution for

spiking in plasma to obtain calibration quality control (QC) samples.

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Preparation of spiked calibration curve standards:

Calibration Curve Standard (CC) Stock Dilution Conc. (ng/mL) Spiked CC Standard Conc. (ng/mL)

Std-H 300000 6000

Std-G 240000 4800

Std-F 120000 2400

Std-E 6000 1200

Std-D 24000 480

Std-C 9600 192

Std-B 3840 76.8

Std-A 1536.7 30.7


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Spiking of plasma for QC samples:

Transfer 0.2ml of each of above stock dilution of moxifloxacin in 10ml volumetric flask and

make up volume with plasma to achieve concentration in following table;

Preparation of moxifloxacin stock solutionfor QC

Amt. Weighed (mg) 1


Potency (%) 99.8


Free Mol wt of compound N/AP

Calculated stock conc.(ng/mL)

1000000

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Table : Moxifloxacin stock dilutions for QC samples

Stock Conc. (ng/ml) Stock Aliquot (ml) Volume made up to(ml)

Final Conc. (ng/ml)

1000000 1.180 5 236000

236000 2.5 5 118000

118000 0.170 5 4012

4012 1.920 5 1540.6

Table : Spiking of plasma for QC samples of Moxifloxacin

Stock Conc. (ng/ml) Final Moxifloxacin conc. in spikedplasma (ng/ml)

Quality control (QC) standards

236000 4720HQC

118000 2360MQC

4012 80.2LQC

1540.6 30.8LOQQC


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Method Development

System Suitability test

System suitability test was performed every day before starting

any method validation exercise. System suitability test of the

LC-MS system was done by giving six injections of referencedilution and %CV for Drug and IS and there retention times

were calculated. The %CV for Drug and IS area ratio should be

within 4% and for retention time should be within 5%.

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Sr.no Moxifloxacin moxifloxacin-d4 Area ratio

Drug Area RT IS Area RT Drug/IS

1 65 0.465 37 0.451 1.7467

2 149243 0.461 101762 0.457 1.4666

3 144898 0.462 99488 0.458 1.4564

4 143113 0.438 98551 0.454 1.4522

5 143242 0.458 100199 0.454 1.4296

6 142652 0.456 95975 0.451 1.4863

7 138404 0.452 94780 0.448 1.4603

8 148 0.448 128 0.448 1.1504

Mean 143592.0 0.455 98459.2 0.452625 1.45857

SD 0.008799 0.003777 0.018552

%CV 1.933923 0.834528 1.30

Acceptance criteria:% CV of mean peak area

ratio

should be 4%% CV of the for retention

times ofdrug and IS

must be 5%

System suitability test

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V lid ti t d


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Validation study Selectivity

Six lots of blank plasma were evaluated and an

interfering peak was observed at the retention time ofthe drug and at the retention time of the internal

standard. The interference at drug retention time in

blank matrix should be 20% of the mean peak area

response of drug in LOQ samples. The interference at ISretention time in blank matrix should be 5% of the

mean peak area response of IS in LOQ samples. The

%CV should be 20% for both drug and IS area in the

LOQ samples.

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Interference at RT of drug should be 20% of mean peak area

response in LOQ Samples

Interference at RT of IS should be 5% of mean peak area

response of IS in all plasma lots

Selectivity

Sr. No Plasma Lots Interferenceat RT of drug

LOQ Arearesponse

Interferenceat RT of Drug(as % of mean

LOQ)

Interferenceat RT of IS

IS Arearesponse

Interferenceat RT of IS( as % of

mean IS area)

1 BLK-01 250 2120 11.9 123 92762 0.14

2 BLK-02 47 1974 2.24 42 91054 0.05

3 BLK-03 96 2154 4.57 45 90552 0.05

4 BLK-04 58 2148 2.76 30 90605 0.03

5 BLK-05 28 2090 1.33 29 90473 0.03

6 BLK-06 11 2119 0.52 0 89990 0.00

Mean 2100.8 90894.3

SD 66.26 984.01

%CV 3.2 1.1

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P i i d A


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Precision and Accuracy:

Precision and accuracy of the method was characterized by running three analytical batches;

each batch contained the following samples:

A reference standard solution (one sample, mixture with internal standard)

Blank matrix

Blank matrix with internal standard

Spiked calibration standards (1 set of 8 non-zero concentrations)

Limit of quantitative quality control (LOQQC) (6samples).

Lower quality control (LQC) (6 samples)

Middle quality control (MQC) (6 samples)

Higher quality control (HQC) (6 samples)

Precision:

The %CV at low , middle and high concentrations should be 15% and at LOQQC should be

20%.

Accuracy:

The mean conc. should be within 15% of the nominal conc. at low, middle, and high QC conc.

and should not deviate by more than 20% at LOQQC conc.

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Within batch precision & accuracy

S.NO LOQQC CONC. LQC CONC MQC CONC. HQC CONC.

1

32.6

75.3

2366.6

4726.5

2

31.0

81.2

2361.8

4721.4

3

31.8

78.4

2358.4

4718.2

4

31.2

69.8

2349.5

4707.2

5

32.2

79.2

2359.4 4719.8

6

31.9

79.3

2357.3 4717.9

Mean

31.78

77.2

2358.83 4718.5

S.D.(+/-) 0.601

4.401

5.640

6.359

C.V.(%)

1.89

5.70

0.23

0.13

Nominal Conc.

30.8

80.2

2360.0

4720.0

% Nominal

103.18

96.25

99.95

99.97


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Interbatch precision & accuracy

S.NO LOQQC CONC. LQC CONC MQC CONC. HQC CONC.

1 32.5 78.2 2449.1 4826.7

2 30.8 81.5 2448.9 4865.1

3 31.3 78.7 2396.3 4956.0

4 32.3 76.02 2380.7 4950.4

5 31.3 76.02 2459.5 4846.7

6 32.3 80.2 2413.5 4918.0

7 31.3 80.2 2389.7 4813.4

8 30.8 79.5 2399.7 4996.7

9 31.5 78.8 2369.9 4773.8

10 32.4 77.2 2424.1 4969.0

11 31.1 74.4 2380.1 5732.3

12 30.7 78.2 2369.2 4189.8

Mean 31.52 78.24 2406.72 4903.15

S.D. (+/-) 0.673 2.04 32.059 337.094

C.V. (%) 2.13 2.60 1.33 6.87

Nominal Conc. 30.8 80.2 2360.0 4720.0

% Nominal 102.33 97.55 101.97 103.88


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Linearity

The linearity of the method was determined by

regression analysis of standard plot associated with a

nine-point standard curve. The calibration curve was

plotted between concentration Vs. area ratio and wasshown to be linear from 30.7 ng/ml to 6000 ng/ml for

Moxifloxacin

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Statistical parameters for calibration curve of Moxifloxacin

Slope, y = 0.00364x + 0.00536

Correlation coefficient, r2 = 0.9991


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Recovery

The percentage recoveries for the drugs and the internal standard were determined by

comparing the peak areas of the response of drug extracted from plasma quality

control samples with that of the peak areas of the unextracted aqueous standard

samples containing the same concentration of the drug and the internal standard..The

recovery is deemed acceptable if C.V. is 20% for %recovery between low, middle

and high quality control concentrations.

The percent recoveries were calculated at each QC concentration by the following

equation.

% Recovery = Mean peak response of extracted samples X 100Mean peak response of non-extracted samples X C.F

C.F.= Concentration of extracted sample

Concentration of non extracted sample

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% Recovery

LQC-Response MQC-Response HQC-Response

S.No. ExtractedSamples UnextractedSamples ExtractedSamples UnextractedSamples ExtractedSamples UnextractedSamples

1 40410.00 51890.00 1394993.00 1958733.00 2494471.00 3442319.00

2 41562.00 54506.00 1374072.00 1908715.00 2730535.00 3396287.00

3 43541.00 56060.00 1460088.00 1948105.00 2661444.00 3477699.00

4 37643.00 55411.00 1386051.00 1940937.00 2628182.00 3552522.00

5 44527.00 58496.00 1411449.00 1941369.00 2683490.00 3548438.00

6 40607.00 57538.00 1382933.00 1872946.00 2770848.00 3592767.00

Mean 41381.67 55650.17 1401597.67 1928512.50 2661495.00 3501672.00

S.D. () 2453.99 2338.20 31335.35 31948.07 96140.68 75199.15

C.V.(%) 4.93 4.20 2.24 1.66 3.61 2.15

% Recovery

74.36

72.68 76.01

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%Total Recovery of Moxifloxacin


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%Total Recovery of Moxifloxacin

Mean 74.36

S.D. () 1.66

C.V.(%) 2.23

% Recovery of Moxifloxacin d-4

S No.Extracted Samples

UnExtracted Samples

1 506373 6545302 476934 668119

3 521707 681840

4 511078 679482

5 516734 6813821

6 510843 686136Mean 507278.16 675238

S.D. () 15788.21 11805.66

C.V.(%) 3.11 1.75

% recovery 75.12


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Stability

Stock solution stability in solvent/solution

Prepared analyte and IS solution and keep aliquots stored at refrigeratedtemperature for specified time period, used as stability stock solution.

Prepared fresh analyte and IS stock solution and used as comparison stock

solution.

Injected six replicate from the vials of the stability stock solution and six

replicate from the vials of comparison stock solution.The %stability should be within range of 85-115%.

The % stability was calculated by the following equation:

% stability= Mean peak area response of stability samples X 100

Mean peak area response of comparison samples X C.F

C.F. = Concentration of stability sample

Concentration of comparison sample

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Stock Solution Stability of Moxifloxacin and Moxifloxacin-d4

S.No. Moxifloxacin Moxifloxacin-d4

Stability stock

Response

Comparison stock

Response

Stability stock

Response

Comparison stock

Response

1 1956733 1923470 683970 671938

2 1905715 1898756 669186 659465

3 1951105 1930876 681660 678438

4 1952937 1926768 681932 677378

5 1938639 1911560 681021 673468

6 1892946 1890678 666430 663762

Mean 1933012.50 1913684.67 677366.50 670741.50

S.D (+/-) 27089.19 16242.87 7520.20 7589.70

C.V. (%) 1.40 0.85 1.11 1.13

(%) Stability 101.00 99.98

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Short term stability

Prepared analyteand ISdilution from the standard stock solutions. Stored at room temperature for 6 to 24 hrs, used as stability dilutions.

Prepared fresh analyte and IS dilution used as comparison dilutions.

Inject 6 replicates of the stability dilutions from the same vial and 6 replicates

of the comparison dilutions from the same vial.

The %stability should be within range of 85-115%.

The % stability was calculated by the following equation:

% Stability = Mean peak area response of stability samples X 100

Mean peak area response of comparison samples

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Short term stability

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Short term stability at room temperature for moxifloxacin and moxifloxacin-d4(IS)

Moxifloxacin Moxifloxacin d-4

S.No. Stability samples

Response

Comparison

samplesResponse

Stability samples

Response

Comparison

samplesResponse

1 535786 539872 548502 551166

2 535092 545413 553470 549400

3 547831 540310 561603 548597

4 544400 531698 558036 544905

5 529916 539053 546695 547416

6 535500 521061 550429 532383Mean 538095.3 536234.5 553122.5 545644.5

S.D (+/-) 6680.07 8635.33 5763.65 6824.37

C.V. (%) 1.24 1.61 1.04 1.25

(%) Stability 100.3 101.37

Freeze-thaw Stability


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Freeze-thaw Stability

Prepared 4 replicates of low and high conc. quality control samples and subject to 3

freeze thaw cycles, used as stability samples.

Each cycle consists of first freezing for at least 24hrs followed by unassisted

thawing at room temperature.

Prepared 4 replicates of fresh low and high conc. quality control samples, used as

comparison samples.

Inject the stability and comparison quality control samples. The %stability should be within range of 85-115%.

The % stability was calculated at each QC concentration by the following equation:

% Stability = Mean peak area response of stability samples X 100

Mean peak area response of comparison samples X C.F

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Freeze thaw stability of Moxifloxacin in human plasma.


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35

Moxifloxacin Stability QC Moxifloxacin Comparison QC

S.No. LQC(ng/ml) HQC(ng/ml) LQC(ng/ml) HQC(ng/ml)

1 78.0 4849.0 77.0 4829.2

2 79.8 4746.8 79.1 4729.2

3 78.8 4794.4 79.6 4737.3

4 76.2 4901.5 79.3 4759.2

Mean 78.20 4822.93 78.75 4763.78

S.D (+/-) 1.523 66.989 1.185 45.402

C.V. (%) 1.9 1.4 1.5 1.0

NominalConc.

96.1 100.7 96.5 99.2

Mean with C.F. 78.56 4752.16

%Stability 99.5% 101.5%

B h t t bilit


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Bench top stability

Bench top stability in matrix

Prepared 4 replicates of low and high conc. quality control samples and stored at

room temperature, used as stability samples.

The storage period should exceed the duration for which the samples maintained

at room temperature in the intended study.

Following the storage period, prepared 4 replicates of fresh low and high conc.

quality control samples, used as comparison samples.

Inject the stability and comparison quality control samples.

The %stability should be within range of 85-115%.

The % stability was calculated at each QC concentration by the followingequation:

% Stability = Mean conc. of stability samples X 100

Mean conc. of comparison samples X C.F

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Bench top Stability in matrix for Moxifloxacin


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37

Moxifloxacin Stability QC Moxifloxacin Comparison QC

S.NO LQC(ng/ml) HQC(ng/ml) LQC(ng/ml) HQC(ng/ml)

1 77.9 4770.6 77.0 4829.2

2 76.1 4844.9 79.1 4729.4

3 76.6 4770.2 79.6 4737.3

4 77.8 4751.7 79.3 4759.2

Mean 77.10 4784.35 78.75 4763.78

Mean with C.F 78.56 4752.16

S.D (+/-) 0.891 41.318 1.185 45.402

C.V. (%) 1.2 0.9 1.5 1

Nominal Conc. 94.7 99.9 96.5 99.2

% Stability 98.1 100.7


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Matrix Factor

A quantitativemeasure of the matrix effect due to suppression or enhancement of ionization

in a mass spectrometric detector is called as matrix factor.

Prepared reference mixture of analyte and internal standard at conc. representing 100%

extraction of analyte and internal standard at low, middle and high QC conc., used as

reference samples.

Processed 6 aliquots from 6 different blank plasma and reconstituted 2 aliquots each with

reference sample at low, middle and high QC conc., used as matrix samples.

Injected these 36 matrix samples along with 4 replicated of reference mixture at

LQC,MQC and HQC level. The %CV of matrix factor between low, middle and high QC

sample should be 15%.

The Matrix factor was calculated at each QC concentration by the following equation:

MF = Mean peak area ratio of matrix sample reconstituted with reference samples X 100

Mean peak area ratio of reference samples

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39

Matrix factor for Moxifloxacin and Moxifloxacin-d4

S.No LQC-Response MQC-Response HQC-Response

(area ratio) (area ratio) (area ratio)

MatrixSamples

ReferenceSamples

MatrixSamples

ReferenceSamples

MatrixSamples

ReferenceSamples

1 0.026 0.0265 0.968 1.0538 2.2061 2.177

2 0.0259 0.0268 1.0011 1.0274 1.9971 2.173

3 0.0266 0.0269 1.0234 1.0359 2.048 2.154

4 0.0246 0.0273 1.0163 1.0697 2.1013 2.117

Mean 0.025 0.026 1.002 1.046 2.043 2.155

S.D. () 0.0008 0.0003 0.0246 0.0188 0.0440 0.0270

C.V. (%) 3.3 1.2 2.5 1.8 2.2 1.3

Matrix factor of Moxifloxacin and Moxifloxacin-d4


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Matrix factor of Moxifloxacin and Moxifloxacin d4

Sample Matrix factor

LQC 1.0

MQC 1.0

HQC 0.9

Mean 0.95

S.D() 0.006

C.V(%) 0.6

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R i j i R d ibili


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Reinjection Reproducibility

Any one of three PA batch analyzed and meeting the acceptance

criteria can be used for establishing re-injection reproducibity.

Re-injected all LOQ,MQC,HQC samples of the selected PA

batch. The % difference of 80% of all QC samples reinjected

should be within 15%.

Calculate the percentage difference for each quality controlsample by the following equation:

% Difference= Absolute (original value-Re-injected value) X100

original value

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Reinjection Reproducibility

O i i l R i j t d % T t l f T t l f % f t t l f l


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Originalvalue

(ng/ml)

Re-injectedvalue

(ng/ml)

%Difference

Total no. ofsampleswhose %differenceare within

15%

Total no. ofsamplesanalysed

% of total no. of sampleswhose %difference are

within 15%

LQC

1 81.1 82.9 2.2 1.248131

2 82.5 79.9 3.2

3 81.5 82.4 1.1

4 77.0 77.8 1.0

5 79.7 81.0 1.6

6 80.2 82.0 2.2

MQC

1 2422.8 2473.0 2.1

2 2469.5 2461.4 0.3

3 2427.2 2455.1 1.1 18 18 100

4 2441.2 2407.1 1.4

5 2421.9 2461.4 1.6


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Conclusion

The developed method for estimation of Moxifloxacin was

validated according to USFDA guideline

All the results obtained were fulfilling the acceptance criteria

The developed method is rapid, sensitive, automated and

specificfor estimation of Moxifloxacin in human plasma.

The method is reliable and can be applied for bioequivalence or

pharmacokinetic study as well as therapeutic drug monitoring

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Key References:

Bressolle, F.; Bromet-pitit, M.; Audran, M. Validation of liquid

chromatography and gas chromatographic methods application

to pharmacokinetics,J. Chromatogr. B. 1996,686, 3-10.

Hartmann,C.; Smeyers-Verbeke, J.; Massart, D. L.; McDowall,

R. D. Validation of bioanalytical chromatographic methods, J.

Pharm. Biomed. Anal. 1998, 17 193-218.

US Food and Drug Administration, Guidance for industry-

Bioanalytical method validation, Center for Drug Evaluation

and Research, Rockville, MD, 2001. (Available at,

http://www.fda.gov/ ) http://www.drugbank.ca/drugs/DB00471

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