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BioAnalytical Technologies (I) Pvt. Ltd.

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Met hod devel opMent &
Va l i da t i on f or
Ma ss spect r oMet r i c
a na l ysi s
b y
N a v na t h Ja yb ha y e
Bi oA na ly t i ca l Technol ogi es ( Ind i a ) Pv t Lt d
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Agenda
Method Development Pathway
Method Optimization for LCMS
New Method Development
Method Validation
Reporting the Sample Analysis Results
References
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Method Optimization for LCMS
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Method Development Pathway
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Four Steps
Mobile Phase : MS- compatible mobile phase
Analytical Column: Adjusting column conditions with
respect to MS
Confirming the MS methods
Sample Preparation Process Optimization
Methods Optimization
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Mobile Phase Requirements
Mobile Phase Solvent
-Volatility, Amount of Water, ionization issues
Mobile Phase Additives
-Volatility, Concentration, ion suppression, buffering capacity
Buffer/pH control
-Formic acid, Ammonium formate, Ammonium acetate, Trifluroacetic
acid
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Mobile Phase Requirements
Mobile phase degassing is an important step in the LC/MS
experiment and can be accomplished via on-line membrane or
vacuum devices, sonication,helium sparging.
Degassing will eliminate pump, cavitation, ensure reproducible
retention times and minimize possible sputering from ion the
source.
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Organic Components
contd
Acetonitrile and Methanol are almost exclusively chosen in LC/MS
method as organic mobile-phase components.
Methanol has:
- Greater phase acidity, polarity, and volatility
than acetonitrile.
- Has been shown 10-50% better sensitivity
than acetonitrile in positive ion mode.
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Limitation there is small chance for methanol analyte adduct
formation
Less frequently used solvent:
Ethanol, 2-Propanol, Tetrahydofluran
Solvent used for Normal Phase Separation:
Toluene, Hexane, Cyclohexane,Dichloromethane and Carbon
Tetrachoride
Organic Components
contd
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Aqueous Components
Careful choice of an appropriate acid or buffer will help ensure
success in the LCMS experiment
Nonvolatile aqueous components greatly decrease and prevent
the detection of ion
Nonvolatile aqueous components can also foul ion sources and
vacuum region of MS
Nonvolatile phosphate and citrate buffer are strongly discouraged
from both ionization and practical reasons
Ion suppression and decreased sensitivity will be observed when
non-volatile buffers are used
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Buffer Consideration:
Phosphate Buffers are commonly used in HPLC because of their
strong buffering capacity and useful pH range unfortunately,
phosphate cannot be used, because they will crystallize out and
contaminate the ion source
Aqueous Components
contd
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Acid and Bases
Formic Acid or Acetic Acid concentration of 0.1 to 1%(V/V) are
recommended in preparing low pH Mobile phases to enhance
ionization in Electrospray
Trifluoro-acetic acid is preferred for protein and peptide
separation, but should be avoided when negative Ion mode is
utilized
Ammonium hydroxide, or, in rarer cases, Triethylamine, are
recommended for high pH mobile phases
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Recommended MP Additives
and Buffers for LCMS
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Isocratic Vs.Gradient Seperation
Isocratic elution: A separation that employs a single solvent or
solvent mixture of constant composition.
Gradient elution: Here two or more solvent systems that differ
significantly in polarity are employed. After elution is begun; the ratio
of the solvents is varied in a programmed way, sometimes
continuously and sometimes in a series of steps. Separation
efficiency is greatly enhanced by gradient elution
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Column Selection & Optimzation
Column selection will based on following requirements:
Chromatographic separation required
Resolution expected
Asymmetric factor
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Analytical Column Optimization
Column selection will based on following information's
Compound Specific information
Nature of compound
Solubility of compound
Polarity of compound
Analytical Column Information
Column Length
Column Chemistry (C8 or C18, )
Physical Properties of column (Pore size, Particle size & diameter)
Sample Specific Information
Origin of Sample
Purity of sample
Sample Preparation technique
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Adjust Column Diameter
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Eliminate Extra-Column
Volume
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Reduce Column Length
150 x 2.1 mm
0.2 mL/min
1500 psi
50 x 2.1 mm
0.2 mL/min
500 psi
0 2 4 6 8 10
Time (min)
0 2 4 6 8 10
Time (min)
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Increase Flow Rate
50 x 2.1 mm
0.2 mL/min
500 psi
50 x 2.1 mm
0.6 mL/min
1500 psi
0 2 4
Time (min)
0 2 4
Time (min)
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Isocratic vs. Gradient Retention
Isocratic: log(k) = log(k
0
) - Su
k* ~
t
G
F
Au V
m
S
Gradient:
Remember: k controls selectivity
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Gradient Separation
In gradient separation, k* controls selectivity.
However, as opposed to isocratic separation, one needs to be
careful to make compensating adjustments to maintain k* if one
of these factors is changed: tG (gradient time), flow rate (F),
gradient range (Df), or column volume (Vm). S is a constant, with
a value of 5 suitable for small molecule calculations. Note that k*
is not influenced by particle size.
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Eliminate Gradient Waste
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Shorten Column
0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8
Time (min)
0 2 4 6 8 10
Time (min)
150 x 4.6 mm, 5 m
1.5 mL/min
15-35%B / 10 min
30 x 4.6 mm, 3 m
1.5 mL/min
15-35%B / 2 min
t
G
F
Au V
m
S
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Reduce Diameter
0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8
Time (min)
30 x 4.6 mm, 3 m
1.5 mL/min
15-35%B / 2 min
30 x 2.1 mm, 3 m
0.3 mL/min
15-35%B / 2 min
1.0 2.0
Time (min)
t
G
F
Au V
m
S
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Other parameter consider in
Method optimization
Define specificity & selectivity
Define LOD & LLOQ
Optimize the sample Preparation Method
Indentify Level of Matrix effect (ME) and system carry over (SCO)
Re-optimization the Method to reduce ME& SCO
Calibration curve building
Pre Method validation Verification
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Summary
Use a volatile mobile phase
Scaling isocratic runs (i.d., length, flow)
Keep k* constant when scaling gradients
Minimize extra-column effects
Beware of dwell volume
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Steps New Method development
Mobile phase selection and Optimization
Analytical column selection and optimization
Detector selection and its parameter optimization
Chromatographic optimization
Define specificity & selectivity
Define LOD & LLOQ
Optimize the sample Preparation Method
Indentify Level of Matrix effect (ME) and system carry over (SCO)
Re-optimization the Method to reduce ME& SCO
Calibration curve building
Pre Method validation Verification
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Method Validation Parameters
Validation of Analytical Method
validation is the process used to confirm that the analytical
procedure employed for a specific test is suitable
for its intended use.
The various validation parameters are:
Accuracy,
Precision (Repeatability and Reproducibility),
Linearity and Range,
Limit of detection (LOD) / Limit of quantitation (LOQ),
Selectivity / Specificity,
Robustness / Ruggedness,
Stability and System suitability studies
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Validation methods
Selective and sensitive analytical methods for the
quantitative determination of drugs and their metabolites
(analytes) are critical for successful performance of PK and
bioequivalence studies.
Validation of analytical methods includes all the procedures
recommended to demonstrate that a particular method, for a
given matrix, is reliable and reproducible
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Validation
methods
1. A prior validation:
Pre-study validation for analytical
method development and method
establishment
2. In-life validation
(Routine validation)
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Regulatory requirements
G.L.P.
(e.g.; bioequivalence, Toxicokinetics)
S.O.P. (standard operating procedure)
(from sample collection to reporting)
Record keeping
Chain of sample custody Sample preparation
Analytical tools
Procedures for quality control and verification of
results
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A prior validation makes sure the method
is suitable for its intended use
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A prior validation: criteria to be validated
1. Calibration curve
2. Accuracy
3. Precision (repeatability, reproducibility)
4. Limit of quantification (LOQ)
5. Limit of detection (LOD)
6. Sensitivity
7. Specificity/selectivity
8. Stability of the analyte in the matrix under study
9. Others (ruggedness, agreement,)
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1. Calibration curve
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Definition
It is the relationship between known
concentrations and experimental response
values
Goal
Determine the unknown concentration of
a sample
Calibration curve
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Response: dependent variable
(peak,area ..)
Y

(
o
b
s
e
r
v
e
d
)
Y
X
y = ax + b
Independent variable:
exactly known
concentrations
Calibration curve Calibration curve
x1
y1
xn
Yn
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Response: dependent variable
Y

(
o
b
s
e
r
v
e
d
)
Y
X
y = ax + b
Independent variable:
X
estimated concentration
^
Calibration curve Calibration curve
x1
y1
xn
Yn
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Calibration
curve
Construction
5 to 8 points over the analytical domain
replicates are required to test linearity
3 to 5 replicates per levels
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Calibration curve
The calibration curve should be prepared in the
same biological matrix (e.g. plasma ) as the
sample in the intended study by spiking with
known concentration of the analyte (or by serial
dilution).
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Reference Standard
Calibration standards and quality control
samples (QC)
Authenticated analytical reference standard
should be used to prepare (separately) solution
of known concentration
certified reference standards
Never from a marketed drug formulation
commercially supplied reference standards
other material of documented purity
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Building the calibration curve:
a regression problem
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Statistical requirements to
build a calibration curve
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Statistical requirements to build a
calibration curve
1. Standard concentration (X) are known without error
2. Variance of response (Y) should be constant over the analytical
domain (homoscedasticity hypothesis); this equivalent to say that
the random errors i are homoscedastic i.e., they all have the
same variance.
3. The random errors i have expected value 0.
4. The random errors i should be independent from Y and are
uncorrelated.
These assumptions imply that least-squares estimates of the
parameters are optimal in a certain sense
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Regression can be used
for prediction
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Regression can be used for prediction
These uses of regression (calibration curve) rely heavily on
the model assumptions being satisfied.
Calibration curve is misused for these purposes where the
appropriate assumptions cannot be verified to hold
The misuse of regression is due to the fact that it take
considerably more knowledge and experience to critique a
model than to fit a model with a software.
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Assessing the calibration curve
the calibration curve (here a statistical model )
should be checked for two different things:
1. Whether the assumptions of least-squares are
fulfilled
Analysis (inspection) of residuals
2. Whether the model is valid and useful
Test of linearity
Back calculations
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Calibration curve : Linearity Calibration curve : Linearity
- Specific tests of linearity should be
used
- The coefficient of correlation (r)
cannot
assess linearity except for r = 1
e.g.: r = 0.999 can be associated with a
calibration curve which is not a straight
line
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R
e
s
p
o
n
s
e

Y
X
Calibration curve: linearity Calibration curve: linearity
Concentration
Test of linearity : Coefficient of correlation
r = 0.99
does not prove linearity
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Freeze/thaw
stability
Avoid freeze and thaw cycles
Enough aliquot samples should be to be
prepared
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Accuracy and precision
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Origin of the error :
Accuracy and
precision
Systematic (not random)
bias
impossible to be corrected
accuracy
Random
can be evaluated by statistics
precision
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Good Precision
Good Accuracy
Poor Precision
Good Accuracy
Good Precision
Poor Accuracy
Poor Precision
Poor Accuracy
Gold
Standard
Silver
Standard
Off-Base
Model
Hit or
Miss Model
Bias and
precision
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Accuracy
Closeness of determined value to the true
value.
The acceptance criteria is mean value s
15% deviation from true value.
At LOQ, 20% deviation is acceptable.
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Accuracy Accuracy
The accuracy is calculated using the following
equation :
Accuracy (%) = 100 x
Found value - Theoretical value
Theoretical value
The accuracy at each concentration level must
be lower than 15% except a LOQ (20%)
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Accuracy
Determination
by replicate analysis of the sample
containing known amount of analyte
5 samples for at least 3 levels
The mean value should be within 15% of the
actual value except at LOQ where it should
not deviate by more than 20%
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Precision
The closeness of replicate determinations of a
sample by an assay.
The acceptance criteria is s 15% CV.
At LOQ, 20% deviation is acceptable.
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Repeatability (r)
Agreement between successive measurements on
the same sample under the same conditions
Reproducibility (R)
The closeness of agreement between results
obtained with the same method under different
conditions
Precision Precision
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PrecisionConsidered at 3 Levels
Repeatability
Intermediate Precision
Reproducibility
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Repeatability
Express the precision under the same
operating conditions over a short interval of
time.
Also referred to as Intra-assay precision
(within day)
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Intermediate Precision
Express within-laboratory variations.
Between days variability
Known as part of Ruggedness in
USP
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Reproducibility
Definition: Ability reproduce data within the
predefined precision
Repeatability test at two different labs
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Precision:
measurement
Should be measured using a minimum of
5 determinations per concentration
A minimum of 3 concentrations in the range
of expected concentrations
The precision at each concentration should
not exceed 15% except for the LOQ (20%)
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Precision:
measurement
for a single measurement : CV(%)
for intra-day and inter-day precision
ANOVA
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Precision: data
analysis
Single level of concentration with repetition
e.g. 12, 13, 12, 14, 13, 14 g/mL
mean : 13.0 g/mL
SD: 0.8944 g/mL
CV% = SD/mean * 100 = 6.88%
CV% is also known as the relative standard deviation or RSD
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Precision: data
analysis
Several levels of concentration and several days
day 1 levels (g/mL) 0.5 5
20
Repetitions 0.4 5.2
20.5
0.5 5.1
21.0
0.4 4.9
19.8
0.6 5.2
18.8
day 2 and 3 : same protocol
ANOVA
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Precision: the statistical
model
The statistical model (for each
concentration level)
Y = + day + c
: general mean
day: an effect (day, technician, or any factor
= inter )
c: error-random = intra
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ANOVA
Allows an estimation of the 2 variance terms
inter-day mean square (BMS)
intra-day mean square (WMS)
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Repeatability and reproducibility
SD for repeatability
o
r
= Var
(e)
SD for reproducibility
o
R
= o
(day) +
o
(r)
variance for reproducibility is the sum of the
variance for repeatability and the inter-day
variance
Inter-day intra-day
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Precision: ANOVA
CV intra: 5%
CV inter: 8%
CV inter > CV intra
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The limit of quantification (LOQ)
LOQ is the lowest amount of analytes
in a sample which can be determined
with defined precision and accuracy
LOQ : 20%
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Limit of quantification (LOQ)
The lowest standard on the calibration
curve is the LOQ if:
no interference is present in the blanks at
retention time of the analyte for this
concentration
the response (analyte peak) has a precision
of 20% and accuracy 80-120%
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Estimation of chromatographic baseline noise Estimation of chromatographic baseline noise
N
p-p
N
p
W : Peak width
1
Baseline
noise
Largest variation
of the baseline
noise
(N )
p-p
Most important
deviation (N )
p
Sample chromatogram
Blanc chromatogram
(a)
(b)
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Three analytical areas Three analytical areas
1 2 3
Xb
not
detected
Area of
detection
Area of
quantification
or CV<20%
LOD LOQ
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The recovery of an analyte in an assay is the detector
response obtained from an amount of the analyte added
to and extracted from the biological matrix, compared to
the detector response obtained for the true concentration
of the pure authentic standard
The recovery allows to determine the percent of lost drug
during sample preparation
Minimal extraction ratio required to ensure a good
repeatability
Recovery: definition
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Absolute recovery is evaluated using low,
medium, and high QC samples and at least three
times for each level
The extraction recovery of the analyte (s) and
internal standard(s) should be higher than 70%,
precise, and reproducible.
Recovery: Determination Recovery: Determination
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Recommended to be a close analog of
the analyte of interest
Advantages and limits
Recovery: Internal standard Recovery: Internal standard
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Recovery
) _ tan , / _ _ _( _
) _ , / _ _ _( _
100 cov Re
solution dard s mL ng x of area Peak
extract plasma mL ng x of area Peak
ery =
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Specificity / Selectivity (1)
Specificity : for an analyte
ability of the method to produce a
response for a single analyte
metabolites
enantiomers
Selectivity: for a matrix
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Specificity / Selectivity (2)
Analyses of blank samples from different subjects (n=6)
Blanks should be tested for interference using the proposed
extraction procedure and other chromatographic conditions
Results should be compared with those obtained with
aqueous solution of the analyte at a concentration near the
LOQ
Blank plasma and pre-dose samples should be without
interference
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Specificity / Selectivity (3)
If more than 10% of the blank samples
exhibit significant interference, the
method should be changed to eliminate
interference
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Definition
The drug must keep all its properties during the
investigations
Stability at room temperature
An experiment should cover 6 to 24h
Stability in frozen biological samples : (-20C or -80C)
Stability sample should allow assay from day 0 to
day 20
Stability during a freeze / thaw cycle
Samples should be frozen and submitted to three
freeze / thaw cycles
Aliquotage is better than repeated freeze / thaw cycles
Stability Stability
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To see this guidance
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To see this
guidance
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