Validasi metoda november 2016 new

Recent development on Validation Method of

Chemical Analysis for (Phyto)Pharmaceutical Preparations

Gunawan Indrayanto

Plant Biotechnology Research Group,

Faculty of Pharmacy, Airlangga University

Surabaya 60286, Indonesi

a

gunawanindrayanto@yahoo.com

This presentation is for Education or Training only;

this presentation is not for commercial purposes

This presentation can not be copied without any permission from the author

Some slides were cited/reproduced from free sources website

Why chemical/Pharmaceutical analysis

is very important?

Chemical and Pharmaceutical Analysis:

•

Pharmaceutical/ Chemical/Food/Cosmetic Industries

•

Diagnostic Laboratories

•

Forensic Laboratories

•

Government Laboratories (BPOM, FDA etc)

•

Government Laboratories (BPOM, FDA etc)

•

Research

How can we have a reliable results of the

chemical analysis?

If the results is NOT valid, all conclusions

will be NOT correct.

Areas

of Chemical Analysis

•

Quantification:

–

How much

of substance X, Y, Z etc is in the

sample?

•

Detection:

–

Does the sample

contain

substance X, Y, Z etc?

–

Does the sample

contain

substance X, Y, Z etc?

•

Identification:

–

What is the

identity

of the substance(S) in the

sample?

•

Separation:

–

How can the species of interest

be separated

from

the sample matrix for better isolation,

quantification and identification?

“Valid”

Methods

Calibrated

Software

?

Reference

Validation

method

compounds

Target compound

must be

stable

in the

Results

of

Analysis

Conclusion(s)

System

Suitability check

Routine Analytical Method

IQ,

OQ, PQ

are valid

Calibrated

Instruments

Qualified

Person

must be

stable

in the

selected solvent

E. Rozet, Ph. Hubert, Presentation University de Liege, Tiblisi, August 2011

Reliable

and

Validated

In order to determine

the “Quality”

of any (Herbal) Drugs,

all chemicals

(API, metabolites, impurities, degradation

products, heavy metals, pesticides, preservatives, sweetener,

toxin) must be evaluated (

qualitatively and quantitatively

).

analysis’s

methods are

needed !

Validation method

for specific compound(s):

drugs, metabolites,

drugs, metabolites,

markers, metals, pesticides,

preservatives

Farmakope Indonesia V

Acceptance criteria of accuracy and

precision are depended

(For compound/marker approach)

depended on Pre-determined Specification Range Or Analytical Target Profile (ATP)

Massart’s Golden rules of Validation method

(Cited by Gonzales & Herrador,

Data Elements Required for Analytical Method Validation

Type of Analytical Procedure

Assay Impurity testing Quantitative Limit tests

Performance charcteristics

Identification

Parameter : USP ICH USP ICH USP ICH USP ICH USP ICH

Yes * *

(For Drugs/Cosmetic/Food analysis)

Accuracy Yes Yes Yes Yes * No * n.a No No

Precision Yes n.a Yes n.a No n.a Yes n.a No n.a

Repeatability n.a _Yes n.a _Yes n.a _No n.a _n.a n.a _No Interm. Prec. n.a _Yes n.a _Yes n.a _No n.a _n.a n.a _No

Specificity Yes Yes Yes Yes Yes Yes * n.a Yes Yes

DL No No No No Yes No * n.a No No

QL No No Yes Yes No No * n.a No No

Linearity Yes Yes Yes Yes No No * n.a No No

Range Yes Yes Yes Yes * No * n.a No No

Step taken for validation Process

Analytical goal

Method selection (Published / new)

Optimization

Stability testing (SST and pre-validation)

Stability testing (SST and pre-validation)

Testing validation parameters

Validation report

Transfer of Method (R&D to QC)

For analysis using (Chromatography)

SST

must be determined

( for standard/ QC samples):

According to USP /39/40 FI V the parameters are:

•Rs (usually > 1.75)

•N

•Tailing Factor ( nicer if ca, 1)

Requirements for doing verification of an official method

•Tailing Factor ( nicer if ca, 1)

•S/N ratio (>5)

•Replicate Injections: see each of monographs or

% RSD (maximal)=

If B= 5, n = 6, max RSD = 2.17 % B = 4, n = 6, max RSD = 1.39 %

Definition of Markers (2008)

:

Not always = maybe “yes”

maybe “no”

Concentrations of

Selected (important) markers could

determine the activities

Must be determined

Stability Studies

Stability Studies

Pre-Validation: Stability testing

•

Scope:

Usage of autosampler for overnight runs with samples

in solution for hours in laboratory environment raises

concern about the stability of samples.

e.g., degradation by hydrolysis, photolysis, adhesion to

glassware, etc.,

•

Recommendations:

•

Recommendations:

Generate data to support standard and sample solution

stability under normal lab conditions for duration of

test procedure, e.g., 24 hours, 3-5 hours for TLC Profile

Acceptance Criteria

Maximal peak area difference is ± 2 %, compare to the

fresh solution

Pre-Validation: Stability data of reference compound and sample should be reported Contoh: Analisa dengan HPLC

Reference standards:

originality (USP, BP, SIGMA, MERCK, etc);

Instrumental analysis (chromatography & spectroscopy

need specific authentic r

standards/compounds

except, if we use Q-NMR (need only general IS)

Purity of the standards

-Using mass Balance Method

Mass balance for primary standard;

(It need (a) calibrated micro/analytical balances)

Smallest minimum weight

Smallest

net weight (w)

according USP: 38 /2015; 39/2016

Sr =

Sr = repeatability ( n = 10) = not more than 0. 10%

W =

Sr = repeatability ( n = 10) = not more than 0. 10%

d = scale interval

For analytical balance d = 0.1 or 0.01mg ; micro-balance 0.001 mg,

w will be = 82 mg , 8.2 mg

,

0.82 mg

(if SD = 0 or , < 0.41 d)

Or w = 2000 SD

USP 35/FI V : Tolerance not exceed 0. 1 % of the amount weight;

so smallest weight must be 100.0 (4 digits)and 10.00 mg (5 digits), or

1.000 mg (6 digits).

Selectivity/Specificity

Specificity/Selectivity

USP, ICH, FDA: Specificity

AOAC, IUPAC : Selectivity

Specificity is

the ability to determine

the analyte unambiguously

in the presence of other components, this includes degradation

products, metabolites, matrix component etc.

products, metabolites, matrix component etc.

Selectivity means that the method provides responses for a

number of chemical entities that may

distinguished

from each

others. It means also

the ability to separate

the target analyte

from all other components

Selectivity evaluation for pharmaceutical preparations

:

Identity of API should be tested, and its selectivity should be

proved.

Please be careful if the identification method that described

in Pharmacopeia/ Official method is not identical with its

in Pharmacopeia/ Official method is not identical with its

assay method (e.g. Identification: IR, color reactions; assay :

HPLC

)

Why for some cases, identification of API in pharmaceutical preparations

should be used by using > 1 methods: IR, TLC, Color reactions

“IR Library” should be validated routine

A B

C R & D/QC Lab. at Pharmaceutical Industry

Nice if related compounds, possible degradation products,

Densitograms (λλλλ = 260 nm) obtained from: (1) solution of standard mometasone furoate, (2) extract from excipients of laboratory-made cream, (3) extract of laboratory-made cream, (4) solution of nipagin, (5) solution of nipasol, (6) extract of commercial ointment-1, (7) extract of commercial lotion, (8) extract of commercial cream-1, (9) extract of commercial ointment-2 and (10) extract of commercial cream-2. Peak identities: (A) mometasone furoate, (B) nipagin and nipasol, (C) unknown.

Wulandari, L, Tan, KS., Indrayanto,G. (2003), J. Liq. Chromatogr. R & T, 26, 109-117

A Standard products, known impurities can be tested

The target peak must be proved regarding its identity and purity

Peaks should be well separated

(F.Melianita, J. Witha, S.Arifin, WF.Karina, G. Indrayanto (2009). J. Liq. Chromatogr. R & T, 32, 567-577)

Rs should be not less 1.75

IF Impurities, related compounds, possible Degradation products

were unavailable

Cited from: D. Widiretani, I. Luailia, G. Indrayanto,

If we have no impurities and/or

degradation products; we should do

stressed

forced experiments to prove the selectivity.

Identity and purity check

of peaks A and B must be performed

Cited from: D. Widiretani, I. Luailia, G. Indrayanto,

J. Planar Chromatogr. 26(2013) , 37-42

Determination of Xanthorrhizol in the rhizomes of Curcuma xanthorrhiza by GC FID

Xanthorrhizol ?

(F. Melinata & G. Indrayanto (2006), Unpublished work)

Should tried

3 different conditions For FID, RID, ELSD (= Blind methods)

Purity Check using PDA detector (HPLC) or Densitometer (TLC-Scanner)

Cited from: M. Yuwono & G. Indrayanto, Profile Drugs Excipients and Related Methodology, Vol. 32, Elsevier, 2005.

M.W. Dong, Modern HPLC

for Practicing Scientist, Wiley, 2006

The purity of the peak can be calculated by

comparing the MS of the u, a and d of the TIC peak.

Acetylsalicylic acid

200 nm

N=12804, Tf=1.20

Evaluation of the shape of acetylsalicylic acid

Evaluation of the shape of acetylsalicylic acid

chromatogram using 5 wavelengths

chromatogram using 5 wavelengths

0 5 10 15(min)

200 nm

220 nm

240 nm

260 nm

280 nm

N=12804, Tf=1.20

N=12970, Tf=1.20

N=12924, Tf=1.20

N=12887, Tf=1.20

N=12924, Tf=1.20

Analysis using LC MS/MS triple Quad

Cited from Agilent LCMSD triple Quad training, Singapore, May 2007

TA Sasaki, Chrom. Tech. July/August 2008

(Mw: 868.04)

M. Distl et al. (2009) Potato Research 52, 39-56

(Mw: 852.07)

The power of MRM

MRM is nicer compare to SIR or SIM

Why we need do the “

Why we need do the “identity test

identity test” and “

” and “

purity test

purity test” of the peaks for

” of the peaks for each

each samples for

samples for

quantitative analysis ?

quantitative analysis ?

•

If the peak is not the target compound,

the conclusion will be not correct.

•

If “impurity components” are included

in the target peak, the quantitative-results

in the target peak, the quantitative-results

will be wrong

•

By Using LC-MS/MS or GC-MS/MS

the target peak doesn’t need to be pure, it can

contain > 1 compounds which have same target

ion;

the Identity of the peaks can be differentiated by using

MS/MS spectrum (but ratio of qualifier to quantifier ions

should be in the acceptable range )

Linearity

•

The linearity of a method is its ability to provide measurement results that are directly proportional to the concentration of the analyte, or are directly proportional after mathematical transformation.

• The linearity is usually documented as the ordinary least squares (OLS) curve, or simply as linear regression curve, of the measured responses (peak area or height) as a function of increasing analyte concentrations

•

Peak area

shall be preferred compared to peak heights for making the calibration curve

• The linearity of the detector can be obtained by diluting of the analyte stock solution, whilst the linearity of the analytical method can be determined by making a series of concentration of the analyte from independent

preparations (weighing, spiking)

(G. Indrayanto, Suciati, M. Yuwono (2010), in Encyclopedia Chromatography 3rd _{Edition,Taylor &Francis, pp.2336-2339)}

•

Analytical Methods Committee, Analyst (1988), 113: 1469-1471.

•

W. Horwitz, Referee (1995), December, 2.

•

J. van Loco; M. Elskens; C. Croux; H. Beernaert. Accred. Qual.

Assur (2002), 7: 281-285

‘Correlation coefficient (r) can not be used alone

for proving the linearity (

r close to 1 is not

a reliable indicator of linearity

)

Assur (2002), 7: 281-285

•

G. Indrayanto; M. Yuwono, in:J. Cazes (ed), Encyclopedia of

Chromatography (Supp.), Marcel Dekker, Inc, New York, NY

10016, 2003.

•

P.Araujo, J.Chromatogr.B. (2009) 877, 2224-2234

•

etc.

Evaluation of linearity/calibration curve

•

Relative process standard deviation (Vxo)- Funk’s

et al. 1992 (< 5 %); “sdv” (CATS from CAMAG) < 5

•Mandel’ test (should be OK)

•Residual test (should be OK)

•ANOVA-linearity testing (should be OK, p < 0.01)

•RSD of the Plot of response factor Vs. concentration (RSD < 2.5 – 5 %) et al. (Xp must be less than the lowest concentration of

•Xp value- Funk’s et al. (Xp must be less than the lowest concentration of the calibration curve)

• % Y intercept < 2

• “r” value (can not be used alone), even “r” is close to one

•Fisher Ratio (according to Araujo 2009)

•G value, Quality Coefficient (According to D Beer et al., 2007)

Some mathematical equations

for evaluation of linearity (Funk’s

et al

., 1992)

(

_{i i}

)

Y xo XO

bx

a

y

y

y

S

S

S

X

S

V

+

=

−

=

=

⋅

=

∑

ˆ

,

ˆ

where

%

100

(

_{i i}

)

Y xo XO

bx

a

y

y

y

S

S

S

X

S

V

+

=

−

=

=

⋅

=

∑

ˆ

,

ˆ

where

%

100

i Y

XO

y

a

bx

N

S

b

S

=

+

−

=

=

,

ˆ

2

where

_{Y i}

XO

y

a

bx

N

S

b

S

=

+

−

=

=

,

ˆ

2

where

(

)

(

)

0.05 p and 2 -N f for factor student t t , 1 1 1 . . Y with . 1 1 . . 2 2 2 2 P 2 2 = = = − = + + + = − + + =

∑

i

∑

i XX XX table Y XX P table XO P X N X Q Q X N t S a Q b Y Y N t S X

The importance of the Xp’s Value for testing the linearity

Xp values must be < X1

Lowest Highest

Testing of Homogeneity (Homoscedasticity)

of the Calibration Curve

Proving Linearity according to Araujo (2009)

– The calibration spots should be independent of each other,

therefore spots should be applied from different, independently

weighed solutions – not by diluting from one and the same

stock solution.

Journal of Planar Chromatography 23 (2010) 3, 173–179 DOI: 10.1556/JPC.23.2010.3.1 173

0933-4173/$ 20.00 © Akadémiai Kiadó, Budapest

stock solution.

– As a consequence, calibration spots should be applied using

uniform volume of solutions, but different concentrations, to

avoid the different band-broadening effects of the different

amounts of solution per spot or band

Working range (WR) of a calibration curve can be defined as:

“the ratio of upper concentration (X

u

)

and

lower concentration (X

l

)”

In QC of Pharmaceutical Industry, WR = 1.5 – 2

Our recommendation: ± 20 % from target concentration ( with n = 4/5)

If WR > 10, It is recommended to use

“a weighting regression calibration curve”

(cited from: S. Kromidas (ed.) , HPLC Made to Measure, Wiley-VCH, Weinheim, 2006,pp. 105-115)

Requirement of assay using Single point Calibration

• Linear response function OK

• Negligible constant systemic error (CL is not significantly different to zero, or

• Negligible constant systemic error (CL is not significantly different to zero, or Intercept < 2 % from target compound )

• Homogeneity of variance

(J. Ermer & P. Nethercote, Validation in PharmaceuticaAnalysis, Wiley-VCH, 2015).

Confidence range of intercept:

Qxx

Xc

N

1

.

S

.

t

a

=

C

2 y 2) -N (P, table

Detection limit (DL), Quantification limit (QL):

•

Impurities

•

Degradation products

•

Cleaning validation

•

Heavy metals

•

Heavy metals

•

Toxic metabolites

•

Pesticides

•

Waste products

•

Bio-analytical methods

Detection Limit & Quantitation Limit

Detection limit (DL) defined as the lowest

concentration of an analyte that can be detected under the analytical condition to be used, but cannot be measured quantitatively.

Quantitation limit (QL) is the lowest concentration that can be determined with acceptable accuracy and precision under the analytical conditions. Generally QL can be estimated as 3 times of DL.

DL and QL for instrumental (chromatographic)

analytical methods can be defined in the term of the signal-noise ratio (2-3/1 for DL and 10/1 for QL)

By constructing a linear regression of relative low concentrations of analyte, accurate value of DL can be calculated, in this case DL = Xp. We

recommend using of 5-10 relatively low

Determination of DL using method of Funk et al (DL = Xa/Xp): QL = 3DL; X2 and X1 (highest and lowest concentrations of standards) should be as low as possible!

Accuracy/ Trueness and Precision

Accuracy/ Trueness and Precision

Evaluation of Accuracy

Accuracy or trueness of an analytical method is given by the extend by which the value obtained deviates from the true value.

Firstly accuracy can be determined by analyzing a sample with known

concentration and comparing between the measured and true value.

The second approach is by comparing test results obtained from new method with results from the existing method that known to be accurate.

The third and fourth approaches are based on the percent recovery of known analyte spiked into blank matrices or products. The last technique is known as standard addition method.

For spiked samples into blank matrices, it is recommended to prepare sample in 5 different concentrations in the level of 80-120% of the target concentration.

For standard addition method, the spiking concentrations are 30-60 % of the label claimed

(Cited from : Indrayanto, G & Yuwono, M. (2003), in : Cazes, J. Ed. Encyclopedia of Chromatography (Marcel Dekker), Supplement

It is not only identification of selected markers (Mars > 3), but should be assayed, and

R must be in the specification range, or Cp/Cpk >1 or BETI < Toxic ?

Final Remark for (QC Herbal) Drugs

• For ensuring the quality of herbal derived drugs a

complete quality control was needed. The content of

selected markers and chromatographic metabolite profile must be both determined and evaluated.

• Authentic reference standards were essential for

determining markers in herbal drugs, whilst for chemical profiling botanical reference materials are chemical profiling botanical reference materials are needed.

• The concentrations of toxic metabolites(s), heavy metals (Hg, Pb, Cd, As), pesticides, mycotoxin

should be below their MPL.

• All methods that will used for QC purposes must be

properly validated before the methods can be routine

THANK YOU