By: Berlian Isnia Fitrasanti

Analytical Toxicology
 Analytical Toxicology
• Detection, identification &
measurement/quantifying foreign component
in biological specimen
• Drug screening lab → protocol: GC/MS or
LC/MS
• Screening → Drug abuse, inorganic substance
 Basic Equipment
• Calibrated and reliable laboratory scale
• Bench-top centrifuge to separate blood
sample and extracted solution
• Vortex mixer or rotary mixer
• Water bath and heating block
• Fridge and freezer
• pH meter
• Automatic and semi-automatic pipette
 Basic Equipment
• Low-power polarised microscope
• Lab glassware
• A supply of chemically pure water
• Compressed air or nitrogen
• TLC plates
• Facility to look at TL chromatogram, including
UV lamp (254 nm dan 366 nm) and fume hood
 Basic Equipment
• UV/Vis single beam or dual beam
spectrophotometer
• Microdifussion Conway dish
• Porcelain spotting tile
• Modified Gutzeit apparatus
 Specimen

Acute Chronic
Blood Sweat

Urine
Hair
Vitreous humor (dead body)
Nail
Skeletal muscle (dead body)

Other tissues (dead body) Bone

• Hair: Baumgartner in 1979 → opiate; 1950s
heavy metal detection.
• Hair → history of drug abuse few months to 3-
5 days before death;
Parietal side is the best → constant growth
• Nail: In 1984, Suzuki et al first time use nail to
detect amphetamine
• Saliva (1950s) → found that substance can be
transported from blood
• Sweat: excretion 2-4L/day
First analysed in 1970
Method: patch/cotton/towel/underwear(has
been done to detect amphetamine)
Can be detected 1-4 weeks after using
• Some drugs appear in sebum in 1-2 hours
after using
• When body fluid is not attainable → skeletal
muscle
• Notes for using hair sample:
– Colour, style (straight/wavy/curly)
– Length
– Amount
– Especially for meth → interaction between
melanin and meth
– Cut hair in every 1-2 cm→ analyse
– Pre-analysis→ should be washed (after cutting)
– Do extraction
 Principle of Analysis

Extraction
• Special for hair • Gas
Chromatography/Mass
• Liquid-liquid Extraction Spectrometry
• Solid Phase Extraction • Liquid
• Solid Phase Micro Chromatography/MS
Extraction
Washing
Analysis
Procedure
HAIR WASHING
Washing/Decontamination Procedure
• Decontamination → erase outside
contaminant on hair; substance amount on
hair < environment contaminant
• 100% decontamination is not possible
• Methanol is frequently used as decontaminant
& substance extraction
• Good washing procedure : Baumgartner & Hill
(used commercially)
 Washing criteria
• Curvature ratio (Rc): ∑drug in 3 pw = 3x(∑drug in
last pw)
• Extended wash ratio (Rew): ∑drug in hair = ∑drug
in last pw
• Safety zone ratio (Rsz): ∑drug in hair = ∑drug in all
pw
• Extended safety zone ratio (Resz): ∑drug in hair=
∑drug in all pw
• New criterion (Rnc): ∑drug in hair = 5x(∑ drug in
last pw)
 Example of decontamination
procedure
• 10 min shampoo & 10 distilled-water rinse; hir
extracted; extracts analysed by RIA
• 15 min ethanol wash; three 30 min pw; hair
digested; digests analysed by RIA
• 15 min IPA wash; three 30 min pw; two 1-h
pw; hair digested, & digests analysed by LC-
MS/MS
Other decontamination procedures
1. 0,1% Sodium Dodecyl Sulphate (NaC12H25SO4)
2. Hair washed with water & methanol (3x) →
ultrasonication (3 min) to decontaminate
3. Dry:
a. RT 3-4 hours; or
b. Heating vial 60C (±1 hour) → dry easier
SAMPLE EXTRACTION
 Principles of Extraction
• Basic drug (80-90%)? Acidic drug (10%)?
Neutral (<10%)?
• Note substance characteristic
• Solubility exchange
– Acidic drugs that are not dissolved in aqueous
solvent & dissolved in ether →deprotonated
– Basic drugs → protonated
– Final results → ion salts
 Specimen Extraction
• First step → drug separation from biological
specimen
• Types of extraction:
– Liquid-liquid Extraction
– Solid-phase Extraction
– Solid-phase Micro Extraction
– Protein precipitation
 Hair Extraction Methods
a. Alkaline method (for drug stabile in alkaline
condition)
2ml 5M NaOH (high concentration) for 4-h→
ultrasonication (brown liquid) → extraction
b. Acidic method (ex: Cocaine, heroin)
1 ml MeOH-HCl (36% HCl) 19:1 + sample → 1-h
ultrasonication → leave overnight → extraction
c. Use buffer/water (meth, cocaine, hypnotic
drugs → recovery )
Shake → crumble → filter → LC/MS
extraction → GC/MS
Metal
Plastic tube
 Liquid-liquid Extraction
• Frequently used as main method
• Expected recovery from extracted drug →
50% or more
• Extraction principles applied in this method
• Frequently used for GC analysis
• pH specimen is customised → extract non-
ionised
 Solid Phase Extraction
• Have been used for years for clinical
toxicology, though rare for postmortem
specimen
• Use polypropylene cartridge
• Results > LLE
• Weakness: Hard to use for postmortem
specimen that has many clots
• Solution: Good IS, good sampel dilution dan
centrifugation
 Solid Phase Micro Extraction
• Does not need complex equipment and
solution
• Can be used for volatile/non-volatile
component in gas and liquid sample
• Consists of fused-silica fibre attached to
stainless steel syringe.
• Directly injected to GC injector for 20-30 min
ANALYSIS
 Substance Analysis
• Volatile substance: Headspace GC
• Non-volatile substance:
– Organic
• Chromatography/Mass Spectrometry
• Immunoassay
– Inorganic → AAS
• Detected based on MW → compared to
IS/Calibration curve
 Thin Layer Chromatography
• TLC made in 1950s
• TLC : most simple method to separate &
identify substance
• Can be used to separate purse substance,
extracted from pharmaceutical formulation,
illegally produced substance and substance in
biological sample
 TLC
• Substance separation on flat surface layered
by stationary phase.
• Mobile phase moves along the surface →
substance will be separated
 Thin Layer
Chromatography

After separation &

seen under UV
lamp
 Gas Chromatography
• GC started to be used in 1950s
• If the substance is volatile enough, so the
molecule can be in gas phase at/under 400C,
& will not disintegrate at this temperature →
GC
• It is necessary to know boiling point of every
substance that will be analysed
Headspace GC
 LC/HPLC
• 1969: HPLC appeared
• 1990s: application of HPLC  → most popular
method of analysis
 LC/HPLC
• Stationary phase → Column
3 types of column:
– C18 (main choice)
– C8
– Phenyl
• Mobile phase
In preparation should use ultra purified water
Ex: 10mM Ammonium acetate dissolved with
purified water → filtrated
 Mass Spectrometry
• Mostly used detector in toxicology analysis
• Advantage of MS:
– Introduce sample to MS
– Sample molecule ionisation
– Separate ionised molecule based on its mass
– Detect and quantify separated ion
– Record, plot and manipulate data
 Mass Spectrometry
• Types of MS:
– Quadrupole
– Ion trap
– Time of Flight
– Magnetic Sector
• Result: Ion separation is carried out based on:
ion mass ratio (m) to ion charge (z) → mass-
to-charge ratio (m/z)
Contoh MS
Hasil MS

SIM
Scan
Analysis Steps

• Method Development
– Extraction method
– Analysis method
• Method Validation
• Sample Analysis
Preparation method
Extraction method
Method Validation
Method Validation

• According to SWGTOX standard

– Bias dan Precision
– Calibration model
– Carryover
– Interference Studies
– Limit of Detection
– Limit of Quantitation
– Dilution Integrity
– Stability
Bias and Precision
• Bias (accuracy) → the difference between the
mean of concentration of measured results and
target concentration of the analytes.
• Precision → closeness of agreement among a
series of measurements obtained from multiple
samplings.
Bias and Precision
• Bias and precision should be done for all quantitative
method
• Assessed by analysing at least 3 different samples per
concentration on 3 different concentration (low, medium
and high) in 5 batch
• Bias data can also be used for precision

• Bias formula:
• Precision formula:

• Max acceptable Bias is 15% for high concentration and 20%

for low concentration
• Max acceptable precision (%CV) is 20%
Example of bias and precision measurement
Intra-day & Inter-day

Intra-day

Inter-day
Calibration model/Linearity

• Calibration model → determined for all

quantitative method → range of analyte
concentration → working range
• Necessary for reliable and accurate results
• 5-8 level of concentration → to see linearity
• Calibration model should be prepared every
day → calculate peak area ratio from an
analyte against internal standard
• Linear correlation coefficient (R2) → > 0.99 →
outlier should be removed
 Methadone
7
y = 0.7722x + 0.009
6
R² = 0.9998
5

PAR
3

0
0 1 2 3 4 5 6 7 8 9
Concentration (mg/kg)

Mirtazapine
2.00
1.80 y = 2.3514x - 0.0233
1.60 R² = 0.9987
1.40
1.20

PAR
1.00
0.80
0.60
0.40
0.20
0.00
0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9
Concentration (mg/kg)

Calibration Curve
Example of outlier
Methadone Muscle 1
8.00

7.00

6.00
y = 0.7127x - 0.0204
R² = 0.9997

5.00
Ratio

4.00

3.00

2.00

1.00

0.00
0 1 2 3 4 5 6 7 8 9
Concentration (mg/kg)
Carryover

• Carryover: Analyte carryover into a

subsequent sample→ inaccurate result
• Carryover evaluation: blank sample is
analysed after high concentration sample →
triplicate analysis
• Highest concentration of calibration model or
higher than that
• If carryover exists → cut-off point or more
wash sample
Interference Studies
• Matrix
– To see any interference from matrix → blood, liver or
muscle
– Analyse blank matrix minimum from 10 different sources
without additional internal standard → no interference
from matrix
• Stable-Isotope Internal Standards
– Blank matrix + IS → signal from analyte of interest
– Interference below LOD → insignificant
• Other commonly encountered analytes
– It is necessary to evaluate other analytes which may be
expected to be present with certain method
– Example: Cocaine method should be evaluated whether
any analyte other than cocaine can interfere analysis
Limit of Detection

• LOD: lowest concentration of analyte that can

be distinguished from noise
• Method of determination:
– Estimating LOD for non-instrumental method
– Using the lowest non-zero calibrator as the LOD
– Using the decision point concentration as the LOD
– Estimating LOD using background noise
• LOD is determined by signal-to-noise ratio (S/N) ≥ 3
Limit of Quantitation

• LOQ: the lowest concentration of an analyte

which meets all identification criteria and can
be quantified with suitable accuracy and
precision
• LLOQ usually > LOD with S/N ratio ≥ 10
• Identification criteria: retention times, target
ion S/N ratio ≥ 10, presence of qualifier ions,
and 3 qualifier ion ratios are met.
• Ex: Amp ions: 190, 118, 91
Dilution Integrity

• Effect sample dilution should be evaluated

• Reason:
– Low specimen volume
– Excessively high concentration (above calibration
range)
• Sample dilution should not affect accuracy
and precision of results
• Repeating bias and precision studies at
common dilution ratios (1:2, 1:10, 1:50)
Recovery

• Definisi: response obtained from an amount

of analyte added to and extracted from a
matrix compared to a response obtained from
true concentration of the pure standard
• Evaluation → 3 replicates of spiked sample at
3 different concentration (low, medium, high)
→ to see the results of extraction method
• Formula:
Stability

• Analyte stability may be affected by many

factors, including storage condition & sample
preparation
• A drug is considered to be stable when there
is no influence of given time and condition on
the contretion of the drug in given matrices
• Instability of target drug → problem
Stability validation method
• Fortified sample of the analytes at low and high
concentration should be prepared
• Fortified samples shall be analysed in triplicate to
establish time zero response
• Freeze/Thaw
– Analyte stability shall be determined after 3 freeze
and thaw cycle
– Analyte is considered stable until average signal
compared to time zero falls outside acceptable bias
• Processed sample
– Evaluate the length of time a processed sample can be
maintained before it undergoes unacceptable changes
(under 3 storage condition: 4°C, -20°C & RT)
Immunoassay
Immunoassay

• Immunoassays have a firm place among

routine methods for the analysis of drugs in
biological fluids and other matrices
• Immunoassay is used to detect whether the
target drug is present
• Immunoassay use an antibody specific for the
drug and labelled form of the same drug/
antibody to generate a measurable signal
Principles of Immunoassay
• RIA:
– popular, high sensitive, able to analyse large number
of samples rapidly and preliminary extraction is not
required
– Antibody-coated tubes → used to good effect w/
iodinated tracers
• Enzyme immunoassay:
– EIA: uses anti-drug antibodies coated onto a
microplate well by passive absorption
– ELISA: similar to EIA, but the anti-drug antibody is
enzyme labelled rather than drug. Drug derivative is
coated onto the plastic well
REPORT
 Report
• Container identification
• Amount of body fluids/tissue
• Colour
• pH test for urine
• Procedure
• Instrument
– Column (type, size)
– Temperature
– Mobile phase
• Result
• Discussion
• Conclusion