3. Method Development
Define your problem
Evaluate your options
Identify potential problem areas
Perform experimental tests-
Start from the detector and work backward.
Keep your experiments simple and well
documented.
Pay attention to the data.
Streamline/automate the method
Characterize the performance
Update and revise the method
4. Perform Experimental Tests
Plan of attack
Choose a technique based on detection limit,
range compound characteristics and
production requirements.
Optimize detector conditions test linearity of
Response, ruggedness, lower quantitation limits
Find/prepare an internal standard homologs,
Similar functional groups and chemical
Properties (stable isotopes for MS/MS!!)
Establish chromatography or other
necessary isolation procedure
9. Optimize detection LC/MS/MS
MS or MS/MS
Choose chemical ionization (heated nebulizer) or electrospray (ionspray).
Positive or negative mode.
If analyte is + ionized in solution => electrospray + mode
If analyte is – ionized in solution => electrospray – mode
If analyte isn’t easily ionized in solutions => chem ionozation
Basic drugs are generally analysed in the + mode
Both chem ionization and electrospray are often applicable,
because most drugs are ionizable in solution.
10. Evaluate the detector issues
Adsorption onto plastic or glass may cause loss of signal
Watch for non-linearity at low concentrations
Avoid MS/MS transitions which are unstable
Loss of water, loss of sodium
Avoid adding ions which interfere with ionization on MS
Non-volatile inorganic salts (phosphate buffer)
TFA causes ion suppression and is retained in source
Ion pairing reagents
TEA can reduce signal, but may be needed for chromatography
Adverse ion source heating effects
Move from heated nebulizer to ion spray
11. Optimize detection LC/MS/MS
Infuse solution mid – high range (1ug/mL) of analyte
Adjust ion source & voltage, to obtain maximum parent ion signal
Choose 2 to 3 fragment ions and optimise voltages then
Fine tune parent- ion combination using temperatures, voltages and
gas pressures
12. Triple Quad Configuration
Q1 Q2 Q3Q0
RF onlyCollision
Cell
Scanning
RF/DC
Scanning
RF/DC
RF only
Q1 and Q3 are standard mass filter quadrupoles.
– The can scan masses sequentially (e.g. 50 to 500 amu)
– The can be used to select a single mass.
Q2 is an RF only quadrupole that is in a gas filled chamber.
– Q2 is the “collision cell” where mass fragmentation occurs.
– Q2 does not filter ions. It accepts all ion sent to it by Q1 and passes
all ions formed by collision to Q3 to be sorted.
13. Multiple Reaction Monitoring (MRM)
Q1 Selects an [M+H]+
Q2 fragments the selected ion.
Q3 monitors only one daughter ion
Q0 Q1 Q2 Q3
N2 CAD Gas
Precursorion
selection
Ion accumulation
Fragmentation
Exit lensStepsMS2: 1 2 3 &4
14. MRM
Only the daughter ion reaches the detector.
Sensitivity of MRM is a function of how much of the
daughter ion is produced.
The parent ion fragmentation to daughter ion is commonly
referred to as a “transition”
Q0 Q1 Q2 Q3
N2 CAD Gas
Precursorion
selection
Ion accumulation
Fragmentation
Exit lensStepsMS2: 1 2 3 &4
17. Perform Experimental Tests
Plan of attack
Work out standard and control
preparation
Isolate the drug and IS from the
matrix- extraction or precipitation
Perform preliminary ruggedness
tests-stability, matrix, dilutions
Perform preliminary performance
tests - accuracy, precision, LOQ, linearity
Consider automation
18. Find the right internal standard
An internal standard is a tool used to normalize all of the
steps performed during sample preparation, separation,
and detection.
An internal standard is important in processing biological
samples because it compensates for losses during
multiple steps of the assay.
It is important to find an internal standard which is
chemically similar to your analyte, and can mimic the
interactions which occur between your analyte and the
biological matrix.
19. Find the right internal standard
What to look for
Similar functional groups
Similar acid base properties (pk values)
Similar polarity and solubility
Similar detector responce
20. Evaluate your options separation/extraction
Separation from
Matrix components – fibrin, proteins, lipids small
molecules (endogenous materials)
Its own metabolites and conjugates
Other exogenous materials-drugs and food
Extraction tools
Liquid-Liquid extraction
Solid phase extraction (off-line or on-line)
Precipitation (from proteins)
Combinations of above
21. Comparison of Extraction Method
Extraction Method Advantages Disadvantages
Protein
Precipitation
•Simple and quick
•Usable for analysis of several analytes
simultaneously
•Solvents used like Methanol, Acetonitrile,
Perchloric Acid
•Non-selective
•Often not suitable for trace
analysis
•Crude extracts can rapidly
block LC column and/or LC-MS
interface
Liquid-liquid
extraction
•Reliable
•Suitable for more extreme pH values in the
extraction matrix
•Often produces ‘clean’ samples
•Labour intensive
•More difficult to automate
•Not suitable for zwitterions and
highly polar compounds
Solid-phase
extraction
•Simple and easily automated
•Can be selective to functional group
interactions
•Wide range of column packing chemistries
available
•Best choice for polar compounds
•SPE cartridge batch variation
resulting in variable recoveries,
sometimes a problem
•Unstable for extreme pHs
which cause deterioration of
some column packing materials
•Blockages possible
•Costly for routine use
21
23. Choose a technique separation/isolation
(HPLC mechanisms)
Reverse phase (RP) – mobile phase is more polar than stationary
phase: more polar molecules elute earlier
Normal phase (NP) - mobile phase is less polar than stationary
phase: less polar molecules elute earlier
Ion-exchange (IE) – interaction between ions of stationary phase and
analyte: more charge, longer retention
Size exclusion chromatography (SEC) – molecules occupy mobile
phase volume depending on ability to partition into pores: smaller
molecules elute first.
24. Finding chromatography conditions
achieved selectively-other conditions
Column dimentions (2to25cm length, 1to 4.6mm id)
Particle type (spherical, irregular, pore size, etc)
End-capping or base deactivation
Temperature of column – increase temperature to
dec Rt while maintaining Rt - 30-400 c is typical
Flow rate
Use of gradient elution
For testing conditions/initial screening
For solving late eluting peak issues
To modify run times to be practical
To enhance sensitivity
25. Finding chromatography conditions
achieved selectively – mobile phase
Tips for mobile phase choice
Choice of solvents may depend on their response at chosen
detector setting. e.g., citric acid, THF and methylene choride
can’t be used below 230 nm.
Adjust pH prior to adding organic
Use volume to volume measures when combining aqueous and
organic solvents.
Density of MeOH/H2O varies with concentration. Can cause high
back pressure.
Degradation of MP on standing can cause drift, particularly TFA
above pH - 2.5 and carbonate buffers.
26. Evaluate the detector lower quantitation
limits
LC/MS/MS
Select “ reasonable” chromatography (k’ 2-3)
1mM formic acid/ACN
Monitor parent and at least 2 primary transitions
Calculate the MQL, maintaining the S/N ratio > 10**
HPLC/UV or Fluorescence
Select “reasonable” chromatography conditions (K’- 3-5)
Using neat (MP) samples, inject low/medium amounts on
column
Calculate the MQL, maitaining the S/N ratio of > 5**
**Guidance page 6- Under final conditions
the SN> 5 and the precision <20%
27. Finding chromatography conditions
process for hplc
Approaches for traditional HPLC
#1Goal in HPLC is resolve all compounds which would have similar Rt and
interfere with detection. That includes analyte, metabolites,
IS < other drugs, and matrix components.
Resolve neat solutions of each analyte and choose internal
standard (s) maintain k’ values of at least 3 for any analyte
Optimize the chromatography for peak shape,resolution, run time(s)
(inject in mobile phase)
Test potential interferences (metabolites, concomitants, endogenous)
Retest on –column MQL of analyte
Choose appropriate concentration of IS based on peak height
28. Finding chromatography conditions
process for LC/MS/MS
Approaches for LC/MS/MS
#1 Goal in LC/MS/MS – to elute the compounds
without having matrix suppression or
enhancement, and having no interferences.
Using mobile phase as injection solvent, test that the Rt of
components of interest which have DIFFERENT transitions, all
have at least 3 k’
Optimise for peak shapes of analytes and internal standard
Re-optimise your detector settings
Check beginning to end run trends for drift
Retest the MQL of the analyte
29. Evaluate the detector test linearity
Detector linearity
Adjust injection amount to avoid column overloading effects
Inject analyte > 2 X the estimation on- column range
Plot ! – response v. amount on column – Evaluate it.
Linearity needs to be established for each analyte
35. One of Many Applications
Surgeons performed the first successful organ transplant of a kidney in 1954.
Since then, it is their dream to prolong life by transplanting organs as a
potentially life saving treatment for end-stage diseases of the kidney, liver,
heart, lung and pancreas. Immunosuppressant drug monitoring plays a
critical role in success of organ transplantation. The pharmacokinetics of
immunosuppressant drugs are complex, unpredictable and prone to
numerous drug interactions with high inter- and intra-individual variability.
They are extensively metabolized (> 25 metabolites of cyclosporine known)
and cross-react with antibodies used in TDM immunoassays. This causes a
significant and unpredictable overestimation of the drug concentrations eg.
Cyclosporin (35–40%). Liquid chromatography–tandem mass spectrometry
(LC-MS/MS) excludes interferences attributable to hydroxylated or
demethylated metabolites of these drugs, thereby providing more accurate
individualized patient dosing and improving the clinical efficacy.
Measurement of immunosuppressants by LC-MS/MS therefore represents
the fundamental work of the clinical chemistry laboratory-Turning blood
into numbers. (Therapeutic drug monitoring by LC-MS/MS- Reema Bahri &
Sreedhara Chaganty – Society of organ transplantation – Indian J. Transplant 2011;
1; 9-45.)
36. References
General extraction/sample preparation
ESP Bouvier et al., LC/GC Intl. Current Trends Developments in
Sample Preparation Step, 1998,35-40.
Precipitation
J. Blanchard, Evaluation of the relative efficacy of various
techniques for deproteinizing plasma sample prior to high-
performance liquid chromatographic analysis. J. Chrom., 226, 455-
460 (1981).
M. Jemal, M. Huang, Z. Jiang, Y. Mao and M.L. Powell, Direct
injection versus liquid-liquid extraction for plasma sample analysis
be HPLC with tandem mass spectrometry. Rapid Column. Mass
Spect. 13, 2125-2132 (1999)
37. References
Solid phase extraction
E. M. Thurman and M.S. Mills, Solid-phase Extraction:
Principles in practice. John Wiley and Sons, NY 1998.
J. S. Fritz, Analytical Solid-phase Extraction. John Wiley
and Sons, NY (1999)
38. References
LC/MS/MS technique
S. Bansal and Z. Liang, “Rapid Unifor and Rational Methods
Development Using LC/MS/MS.” Paper presented at the 46th
Amer. Soc. For MSC, Orlando, Fl, 31 May-4 June 1998.
J. Henion, E. Brewer, and G. Rule, LC/MS Sample preparation
Chemist at Work, 8/2, 36-42 (1998)
E. Brewer and J. Henion, Atmospheric pressure ionization
LC/MS/MS techniques for drug disposition studies.
J. Pharm. Sci. 87 (4) 395-402 (1998)
40. Find the right internal standard
Where to look
The chemist who made the analyte
Medicinal chemistry and pharmacology texts
Lists of compounds with similar characteristics (pk,
chromatographic, spectral)
Synthesize it – expects 1 to 3 months delay ($10 to
$40k)
41. Find the right internal standard
What are some issues?
Can make quantitation worse, particularly in MS/MS
May need more than one if there are multiple analytes of different
characteristics.
Increases run time if elutes late
Should be readily available for extended development programs
and method transferability.
COA good to have- not required- but has been subject of a 483.
42. Find the right internal standard
Suggestions
LC (traditional) – elute after the analyte to avoid
polar metabolites
Adjust on-column amount to mid-range
Regularly monitor its response throughout runs
Use a stable isotope for LC/MS/MS wherever possible
43. Sample extraction fundamental objectives
Identify functional groups on analyte that influence its
solubility, polarity, etc.
Understand how an analyte behaves in response to changing
extraction conditions (e.g., solution pH, % organic)
Manipulate these conditions to meet defined method
objectives
44. Sample extraction precipitation
(be suspicious!)
When to use
Simple matrices - e.g., Urine
Small total number of samples
Concentrations > 500ng/mL
If there is low protein binding
With column switching or secondary cleanup procedure
45. Sample precipitation – typical method
25 to 100ul sample
100 to 500uL, MeOH 25uL
TCA, 6%HClO4, or 10% Zn or Cu salt
14000 g possible
Causes dilution, may need to evaporate
first. Solvent needs to be similar to
MP
Transfer sample,
standard or control
Add precipitant with IS
vortex
Adjust solvent and inject
Centrifuge at high speed
Transfer supernatent
to autoinjection vial
46. Sample extraction precipitation
Advantage of precipitation
Fast, cheap, simple
Separation from majority of proteins
Generally high recovery of analyte
Fast method development
47. Sample extraction precipitation
Disadvantages of precipitation
Little/no cleanup from similar analytes or polar molecules
Matrix differences can cause significant issues (blood, tissues)
Excess materials can clog up column or decrease lifetime
Need to get sample back into mobile phase
Potential binding to proteins - loss of recovery.
Limited sample volume
Minimal concentration effect
48. Sample extraction solid-phase
extraction
First line of experiments for most small molecules
In SPE, a solid support packing with sorbent material is packed in
a small column. The analyte is retained on the bed as potential
interferences are washed off. After 1 to 3 washings of different
solvent strength, the column conditions are changed to allow the
analyte to be eluted and collected.
49. Sample extraction solidphase
extraction- typical procedure
Adjust pH and organic content to
retain analyte
100 uL to 1.0 mL samples. Use
appropriate mobile phase to keep
all of analyte on column.
Use varying strength and pH to
remove interferences
Use vacuum, pressure or centrifuge
Try to match mobile phase
Condition Column
Add buffer and internal
standard to sample
Elute
Apply sample to column
Wash 1 to 3 times
Adjust organic in final solution
Inject
50. Sample extraction
solid phase extraction
Advantages of SPE
Fast method development
Wide variety of options of columns
Useful for many different types of analytes
Very automatable
Disadvantages of SPE
Expensive
Difficult to concentrate sample
Similar analytes may not be separated
Need to get sample back into – mobile phase
Potential binding to proteins - loss of recovery.
52. Sample extraction
Liquid - Liquid extraction
When to use
Best clean-up desired
Expect many sample over a long time period
Difficulty removing interferences
Issues with ion suppression on MS
When derivatization is desirable
53. Sample extraction
Liquid- Liquid extraction
Up to 2mL
(Base) use 2 pH units > pKa to
suppress ionization
Ether , CHCl3 , hexanes, combos
pH > 2 units
below PKa (bases)
Sample in screw cap tube
Add buffer and internal standard
Add non-polar organic
Shake or vortex, centrifuge
Remove organic and dry Add aqueous solvent
Adjust to MP and inject
54. Sample extraction
Liquid- Liquid extraction
Advantages of LLE
Wide variety of conditions
Useful for many different types of analytes
Easy to concentrate sample
Useable for large sample volumes to maximize LLOQ
Good removal from proteins and many polar molecules
Semi-automatable
55. Sample extraction
Liquid- Liquid extraction
Disadvantages of LLE
Method development slow
Recovery in first extraction may be low
Multiple steps
Uses organic solvents.
57. Value of Bioanalytical Validation
Should we validate a bioanalytical method is
not a question anymore...
Validation proves the quality of the analyst’s work
Users of bioanalytical data get confidence in the results
It is required by the regulatory agencies,
Uniformity and a harmonized view on validation would
help all bioanalysts
59. Accuracy and Precision
Determined by RE% and
CV% (based on nominal
concentrations), other
statistical methods are
acceptable
Min. of 5 determinations
per concentration level
Intra-day and Inter-day
Inter-day: 3-6 days?
(depending on assay
variability)
Calibration standards
6-8 non-zero standards
±20% at LLOQ
±15% for all other concentrations
At least 75% passing
Quality Control Samples
Min. of 6 at 3 concentrations (low,
mid, high) or 5% of unknowns
At least 4 out of 6 passing, and at
least one at each conc.
±15% for all concentrations
60. Selectivity
Evidence that you are measuring the intended analyte
Prove non-interference from matrix by using a min. of 6
independent sources of the same matrix
Selectivity should be ensured at LLOQ
For LC/MS methods 6 matrices not required, but must investigate
matrix effects
61. Sensitivity
Lowest concentration on standard curve that can be measured
with acceptable precision and accuracy
Determined using 5 samples-independent of standards
Should not be confused with the limit of detection (LOD) of
the assay
62. Reproducibility
Re-injection reproducibility – To determine if a run could be
reanalyzed in case of delays
Inter-day precision and accuracy is a measure of reproducibility
Freeze/thaw cycles
Repeat analysis of incurred samples (freeze/thaw) to avoid
surprises later and prove reproducibility of unknown concentrations
63. Stability
Stability of analyte in the matrix during collection and storage of
samples should be assessed
Stock solution (min. 6 hr.), freeze/thaw, RT (4-24 hr), extracted
samples, long term stability
Additional stability from incurred samples
64. Response Function
Standards in the same matrix as samples
Sufficient no. of points to adequately define conc./response
relationship
6-8 points, blank, blank + IS
Relationship should be continuous and reproducible and use
simplest model for curve fitting with weighting
65. Extraction Efficiency
Determine at 3 concentrations, Low, Mid and High
Recovery should be consistent, precise and reproducible
Does not need to be very high, but must be consistent
66. Partial Validations
Now allowed in regulatory guidance…
Method transfer between labs.
Change in matrix within species
Change in species within matrix (excellent for LC/MS/MS
methods)
Rare matrices
Take the max. use from these, as these are scientifically valid
67. Cross Validation
When samples within a single study are analyzed at more than
one site or lab.
When different analytical techniques are used in different
studies
Should be done with spiked samples and incurred samples
68. Documentation for Bioanalytical Data
New requirements in the guidance document
Summary tables
Method development and establishment (validation)
BA reports for samples analysis (Include 5-20%
chromatograms full with stds and QCs)
Other information related to 2 and 3 (abbrev., codes, references,
SOPs)
