CENTER FOR BIOEQUIVALENCE STUDIES AND BIOASSAY RESEARCH
DR. PANJWANI CENTER FOR MOLECULAR MEDICINE AND DRUG RESEARCH
INTERNATIONAL CENTER FOR CHEMICAL AND BIOLOGICAL SCIENCES
UNIVERSITY OF KARACHI
ANALYTICAL PHASE OF
BIOEQUIVALENCE/PHARMACOKINETISC
STUDIES
DR. NAGHMA HASHMI
CENTER FOR BIOEQUIVALENCE STUDIES AND BIOASSAY
RESEARCH (CBSBR)
CENTER FOR BIOEQUIVALENCE STUDIES AND BIOASSAY RESEARCH
DR. PANJWANI CENTER FOR MOLECULAR MEDICINE AND DRUG RESEARCH
INTERNATIONAL CENTER FOR CHEMICAL AND BIOLOGICAL SCIENCES
UNIVERSITY OF KARACHI
Analytical Phase the most critical, most crucial and most decisive
comprises of :
 Development of Bioanalytical Method
 Validation Of Bioanalytical Method
 Biological Sample Analysis
ANALYTICAL PROCEDURES
Method Development
Method Validation
Analysis of Biological sample
CENTER FOR BIOEQUIVALENCE STUDIES AND BIOASSAY RESEARCH
DR. PANJWANI CENTER FOR MOLECULAR MEDICINE AND DRUG RESEARCH
INTERNATIONAL CENTER FOR CHEMICAL AND BIOLOGICAL SCIENCES
UNIVERSITY OF KARACHI
TYPE OF ANALYTE
(Parent vs. Metabolite)
Generally parent drug.
 Metabolite acceptable ‘in exceptional cases’
 Present any available data supporting the view that the
metabolite exposure will reflect parent drug.
Metabolite formation is not saturated at therapeutic doses
CENTER FOR BIOEQUIVALENCE STUDIES AND BIOASSAY RESEARCH
DR. PANJWANI CENTER FOR MOLECULAR MEDICINE AND DRUG RESEARCH
INTERNATIONAL CENTER FOR CHEMICAL AND BIOLOGICAL SCIENCES
UNIVERSITY OF KARACHI
Basics of Bioanalytics
Biological matrices
Whole Blood
Serum
Plasma
Urine
Liquor, Saliva, Synovia, Tissue, Faeces, Sputum,…
CENTER FOR BIOEQUIVALENCE STUDIES AND BIOASSAY RESEARCH
DR. PANJWANI CENTER FOR MOLECULAR MEDICINE AND DRUG RESEARCH
INTERNATIONAL CENTER FOR CHEMICAL AND BIOLOGICAL SCIENCES
UNIVERSITY OF KARACHI
Biological matrices
Whole Blood
Should be avoided if ever possible…
Mandatory if drug binds strongly to erythrocytes (i.e., no plasma
concentrations can be measured – example: chlorthalidone).
Recommended if drug is routinely in Therapeutic Drug Monitoring
(TDM) in whole blood, even if measurement in plasma is possible.
Examples: tacrolimus; ciclosporin
CENTER FOR BIOEQUIVALENCE STUDIES AND BIOASSAY RESEARCH
DR. PANJWANI CENTER FOR MOLECULAR MEDICINE AND DRUG RESEARCH
INTERNATIONAL CENTER FOR CHEMICAL AND BIOLOGICAL SCIENCES
UNIVERSITY OF KARACHI
Biological matrices
Serum
Only if no stability issues (sufficient time for clotting– no cooling
allowed).
Possible problems after thawing (turbidity requiring centrifugation
Sometimes problems become evident only in multiple freeze-thawcycles (part of validation, but not of method development).
Therefore most bioanalysts opt for…
Plasma
Better choice than serum,
Numerous anticoagulants available: heparin, citrate, different EDTA
salts (Na, K, Li).
Type of anticoagulant must not interfere with the extraction / separation
/ detection!
It is the responsibility of the bioanalyst to require the most suitable
anticoagulant from the clinical site.
Urine, Liquor, Saliva, Synovia, Tissue, Faeces, Sputum,…
CENTER FOR BIOEQUIVALENCE STUDIES AND BIOASSAY RESEARCH
DR. PANJWANI CENTER FOR MOLECULAR MEDICINE AND DRUG RESEARCH
INTERNATIONAL CENTER FOR CHEMICAL AND BIOLOGICAL SCIENCES
UNIVERSITY OF KARACHI
Sample Preparation
(Extraction / Trace Enrichment)
Dilution
Protein Precipitation
Liquid-Liquid Extraction (LLE)
Solid Phase Extraction (SPE)
Dilution
For urine samples – with a few exceptions
Protein Precipitation
Rough treatments’ (perchloric acid, trichoroacetic acid) should
be
avoided: analyte will be trapped in denaturized protein clots.
acetonitrile, methanol or ethanol is recommended specially for LC/MS
Best choice for high concentrations, good separation and
selective detection
All other low molecular mass compounds (incl. endogenous
ones) remain in solution.
Led to many disasters in LC/MS-MS (matrix effects)!
CENTER FOR BIOEQUIVALENCE STUDIES AND BIOASSAY RESEARCH
DR. PANJWANI CENTER FOR MOLECULAR MEDICINE AND DRUG RESEARCH
INTERNATIONAL CENTER FOR CHEMICAL AND BIOLOGICAL SCIENCES
UNIVERSITY OF KARACHI
Sample Preparation
Liquid-Liquid Extraction
Based on distribution between an aqueous phase (plasma, urine) and an organic
phase Simple example: diclofenac.
Solid Phase Extraction
Based on distribution between an aqueous phase (plasma, urine) and chemically
bonded solid phase matrix.
Most common reversed phase silica: C18, C8, C4, C2, Phenyl
Rarely normal phase: NH2, OH, CN
At least for BE studies – free (unbound) drug should be measured, even for
highly protein bound drugs. Common sample preparation techniques lead always
to the total concentration.
CENTER FOR BIOEQUIVALENCE STUDIES AND BIOASSAY RESEARCH
DR. PANJWANI CENTER FOR MOLECULAR MEDICINE AND DRUG RESEARCH
INTERNATIONAL CENTER FOR CHEMICAL AND BIOLOGICAL SCIENCES
UNIVERSITY OF KARACHI
Separation Techniques in Bioanalytics
for ‘Small Molecules’: Chromatographic Method
Gas Chromatography (GC)
 High Performance Liquid Chromatography (HPLC/UHPLC)
 High Performance Thin Layer Chromatography (HPTLC)
Capillary Electrophoresis (CE)
 Supercritical Fluid Chromatography (SFC)
 Hyphenated Techniques(GC/GC, LC/LC, SFC/GC)MS-MS, TOFMS
for Biologics: Ligand Binding Assays
Enzyme Linked Immunosorbent Assay (ELISA),
Enzyme Multiplied Immunoassay Technique (EMIT)
Radioimmunoassay (RIA)
CENTER FOR BIOEQUIVALENCE STUDIES AND BIOASSAY RESEARCH
DR. PANJWANI CENTER FOR MOLECULAR MEDICINE AND DRUG RESEARCH
INTERNATIONAL CENTER FOR CHEMICAL AND BIOLOGICAL SCIENCES
UNIVERSITY OF KARACHI
Detection
for GC
Flame Ionization Detection (FID)
 Nitrogen-Phosporus Detector (NPD)
 Electron Capture Dector (ECD)
Mass Spectrometry (MS)
 Thermal Conductivity (TCD)
for HPLC
Ultraviolet / Visible (UV/Vis), Diode-Array (DAD) Fluorescence (FL)
Electrochemical (EC; coloumetric)
Mass Spectrometry (MS)
 Conductivity, Diffraction (Evaporative Light Scattering (ELS),
Refractive Index (RI), Radioactivity (RA),…
CENTER FOR BIOEQUIVALENCE STUDIES AND BIOASSAY RESEARCH
DR. PANJWANI CENTER FOR MOLECULAR MEDICINE AND DRUG RESEARCH
INTERNATIONAL CENTER FOR CHEMICAL AND BIOLOGICAL SCIENCES
UNIVERSITY OF KARACHI
Integration
Peak ‘recognition’ Peak start and end ‘recognized’ by:
Noise threshold
Baseline drift: mainly important for gradient elution
 Area threshold: values below this value are not followed
Automatic vs. manual The automatic integration may fail:
Mainly for small peaks close to the LLOQ
Rarely for high peaks, when a series of positive random noise may
trigger a ‘end of peak’ too early or negative random noise draws the
baseline too late.
Chromatography Data Systems (CDS) Chromatography Data System (CDS)
Bundled with chromatograph / MS
Xcalibur® (Thermo Scientific)
Analyst® (Applied Biosystems/MDS Sciex)
EZChrome Elite (Agilent Technologies)
Empower™ (Waters)
LabSolutions (Shimadzu)
CENTER FOR BIOEQUIVALENCE STUDIES AND BIOASSAY RESEARCH
DR. PANJWANI CENTER FOR MOLECULAR MEDICINE AND DRUG RESEARCH
INTERNATIONAL CENTER FOR CHEMICAL AND BIOLOGICAL SCIENCES
UNIVERSITY OF KARACHI
Calibration
Model Selection
Linear y = A + Bx
Quadratic y = A + Bx + Cx²
4-parameter logistic (LBA)
Weighting Schemes
Unweighted (simple linear regression; not
recommended)
Weighted w=1/x, w=1/x², w=1/s²
Working Range
accurate and precise within Lower Limit of Quantitation (LLOQ) and Upper
Limit of Quantitation (ULOQ)
CENTER FOR BIOEQUIVALENCE STUDIES AND BIOASSAY RESEARCH
DR. PANJWANI CENTER FOR MOLECULAR MEDICINE AND DRUG RESEARCH
INTERNATIONAL CENTER FOR CHEMICAL AND BIOLOGICAL SCIENCES
UNIVERSITY OF KARACHI
Considerations
Absence of co-eluting compounds (MS-MS < MS < FL < EC < UV-DAD < UV)
Lack of Matrix Effects
peak response in presence of matrix ions
MF= ___________________________________
peak response in mobile phase
Matrix Factor ~1
Minimization of Matrix Effects
Use internal standard of similar structure (preferably stable isotope –
labeled; recommended in EMA 2011 GL)
Avoid ‘precipitate and shot’–methods Conduct sufficient sample cleanup.
Use new chromatographic methods (UPLC, Rapid Resolution LC) to
enhance separation
CENTER FOR BIOEQUIVALENCE STUDIES AND BIOASSAY RESEARCH
DR. PANJWANI CENTER FOR MOLECULAR MEDICINE AND DRUG RESEARCH
INTERNATIONAL CENTER FOR CHEMICAL AND BIOLOGICAL SCIENCES
UNIVERSITY OF KARACHI
Capacity factor k’ for analytes >2
Example:
(6.58–1.50)/1.50=3.39
(8.46–1.50)/1.50=4.64
Resolution between two adjacent peaks >2
Rs = 2 × (tR2 – tR1) / (w1 + w2)
Example: 2×(8.46–6.58)/(0.68+0.83)=5.69
Tailing factor Tf for analytes <2
t
CENTER FOR BIOEQUIVALENCE STUDIES AND BIOASSAY RESEARCH
DR. PANJWANI CENTER FOR MOLECULAR MEDICINE AND DRUG RESEARCH
INTERNATIONAL CENTER FOR CHEMICAL AND BIOLOGICAL SCIENCES
UNIVERSITY OF KARACHI
Method Validation
Selectivity
Recovery
Linearity
Ruggedness
Accuracy & precision
Intra-Day Accuracy and Precision
Inter-Day Accuracy and Precision
Stability
Auto-Sampler Stability
 Freeze Thaw Stability
 Long Term Stability
 Stock Solution Stability
CENTER FOR BIOEQUIVALENCE STUDIES AND BIOASSAY RESEARCH
DR. PANJWANI CENTER FOR MOLECULAR MEDICINE AND DRUG RESEARCH
INTERNATIONAL CENTER FOR CHEMICAL AND BIOLOGICAL SCIENCES
UNIVERSITY OF KARACHI
Analysis of Biological Sample
Calibrators Full range of calibrators covering Lower Limit of Quantitation (LLOQ)
and Upper Limit of Quantitation (ULOQ)
2.5
2
1.5
Area Ratio
1
0.5
0
R² = 0.9863
0
50
100
Conc.
Quality Control Samples QCL , QCM , QCH
Volunteer Samples zero and 0.25, 0.5, 0.75,1.0, 1.5, 2.0, 2.5, 3.0, 3.5,4.0, 6.0,
8.0, 10.0, 12.0, 24, 36 and 48 (18 Time Points)
Period I + Period II = 36 ( Total number of samples)
CENTER FOR BIOEQUIVALENCE STUDIES AND BIOASSAY RESEARCH
DR. PANJWANI CENTER FOR MOLECULAR MEDICINE AND DRUG RESEARCH
INTERNATIONAL CENTER FOR CHEMICAL AND BIOLOGICAL SCIENCES
UNIVERSITY OF KARACHI
Analysis of Biological Sample
Sample code
193
194
195
196
197
198
199
200
201
202
203
204
205
206
207
208
Lot # 01 (09-Sep-13)
drug area
IS Area
area ratio
0
72611
59459
56182
51414
43429
38459
29143
18632
15432
11269
10988
8876
44174
3127
3069
22739
22541
20253
21178
19595
21193
20798
21965
21383
23209
22924
22501
21895
24341
21304
21913
0
3.22128566
2.93581198
2.65284729
2.62383261
2.04921436
1.84916819
1.32679262
0.8713464
0.66491447
0.49158088
0.48833385
0.40538936
1.81479808
0.14677995
0.14005385
measured conc.
0.000
126.207
114.750
103.393
102.228
79.165
71.136
50.170
31.890
23.604
16.647
16.517
13.188
69.756
2.808
2.539
CENTER FOR BIOEQUIVALENCE STUDIES AND BIOASSAY RESEARCH
DR. PANJWANI CENTER FOR MOLECULAR MEDICINE AND DRUG RESEARCH
INTERNATIONAL CENTER FOR CHEMICAL AND BIOLOGICAL SCIENCES
UNIVERSITY OF KARACHI
THANK YOU