Analytical Forensic Toxicology Exam 2

GCMS, Derivatization, LLE

internal standard addition

always after dilution to ensure same amount in each sample

internal standard properties

mirror analyte behavior, not already present in sample, can be separated via chromatogram or mass, does not react with analyte, soluble and stable in diluent, accurately added to samples

how to choose a method

AMR chosen and capability of instrument

AMR

analytical measured range

AMR def

range you can quantitate and report a value based on method validation

internal standard purpose

verification, identification, quantitation

internal standard verification

deemed by successful extraction

internal standard identification

identify compound through retention time comparison 

internal standard quantitation

calibration curve

verify before report

calibration % dev, controls in range, negative blanks, no carryover, ion ratios, itsd recover, retention time, rrt, chromatography

internal standard for non specific detector

similar structure, not found in sample, no shared common ion, same extraction, acceptable chromatogram 

internal standard for mass spec

isotopically labeled version if analyte or matched

matched isotope 

adding deuterium

internal standard accounts for 

any loss in sample

standard addition

generate quant results within sample instead of performing external

standard addition common use

no validated method to run sample, difficult matrix like liver tissues

low pka=

stronger acid=donates proton more

pH variable dependent on 

matrix and/or extraction solvent

ionization formation ideal

2 pH units away from pKa

amphoteric compounds

has both acidic and basic groups, act as either

zwitterion

has both a pos and neg charge at the same time

direct analysis sample prep types

enzyme immunoassay, headspace

dilute and shoot pros

simple, cheap, easy, reduce viscosity

dilute and shoot cons

potential loss of recovery by diluting analyte, doesn’t remove contaminants,

protein precipitation

dilution and take out matrix components by denaturing proteins

protein precipitation con

doesn’t take out chemical contaminants/interference

partition coefficient 

tendency for any species to prefer to be in one chemical phase over another after equilibrium

partition coefficient formula

conc in phase 2/ conc in phase 1

partition coefficient for high recovery

as high and as different as possible

types of extraction 

LLE, SPE, SLE, PLD

LLE

liquid liquid extraction

SPE

solid phase extraction

SPE process

sample enters syringe column bed, different liquids are passed through to elute desired target or just the analyte

SPE pros

selectivity, flexibility, high automation potential

SLE

supported liquid extraction

SLE process

one liquid flows through solid support in column trapping some compounds, extracting liquid flows through taking target to collection

PLD

phospholipid depletion

LLE steps

strong base convert analyte, analyte leaves aqueous, mixing and separating layers, isolate layer with compound, run on instrument or dry down

LLE affected by

pH, temperature, affinity for solvent, affinity for matrix, relative volumes, number of extraction steps, immiscibility of phases

LLE three aspects to consider

matrices, the solvents, the analyte

LLE back extraction use

3 layer to increase purity 

LLE back extraction layers

add base to neutralize and migrate organic, add acid to attract to aqueous, add base

LLE cons

limited selectivity, difficulty of automation, emulsions

LLE pros

rapid method development, simplicity

emulsions

sample contains high level surfactants that prevent clean separation

fixing emulsions

sample sit, acidify the sample, add table sat, filter sodium sulfate, breakup and centrifuge, ultrasonic bath, change the extraction type

derivatization limitation

can make the molecule too large to volatize 

derivatization requirements

heat, time, catalyst, removal of reactant 

derivatization goal

chemically react with problematic molecule in native form and convert to gc acceptable form 

derivatization outcomes

improve chromatogram, differentiate optical isomers, create charge for LCMS

GC pros

most successful chromatogram, cheap, robust

gc main contaminants

oxygen, moisture, hydrocarbon 

GC process

cold sample passes through hot inlet and condenses onto column, oven heats column to provide movement with carrier gas through to detector

septum purge

tiny flow rate to blow away debris

Splitless flow

slow rate, goes through liner, everything onto column

splitless cons

broader bands (higher B), more time for anaytes to breakdown or adsorb 

split injection liner

tapered liner

split flow

high rate, takes sample with gas to split vent

split pros

larger quantities, preserve column capacity

split ratio

sample waste: sample to column, high ratio= lower amount of sample enters

split flow peak affect

high flow= sharper peaks 

on column use

thermally labile compounds (can’t be heated/explosive)

on column flow and temp

slow flow rate, inlet temp=same as column

on column requirements

specific syringe, wider bore column

Headspace injection types

purge and trap, SPME

SPME

solid phase micro extractions

purge and trap process

push gas into sample, force gas out to be collected in trap, put onto column after a bit

SPME process

sample absorbs into needle/fiber from liquid or gas, fiber introduced to injection port

SPME pros

minimal sample prep, no solvent, different fibers

heated oven purpose

maintain precise control over column temp and changes

Types of oven temp

isothermal, program, zones, ranges, design

oven temperature affects

volatility and flow rate through the column 

Chromatography theory

retention dictated by the relative amount of time spent in the stationary phase compared to mobile phase

low affinity to stationary elution effect

short elution, low retention time

system prevention of band broadening

column fittings to inlet and detector reduce dead volume to minimal levels

column efficiency

relative measure of ability to generate clearly resolved peaks

More theoretical plates means

more separation= better resolution

longitudinal diffusion on flow rate

small diffusion= increase flow rate

mass transfer

time analyte equilibrates between two phases before moving to the next plate

GC parameters

temperature, gas flow, carrier gas, injection port liner, injection mode, detector, acquisition

column dimensions

length, diameter, film thickness, temperature, stationary phase

small internal diameter pro

highest efficiency, shorter time, highly complex samples

longer column pro

high resolution

longer column cons

too much time, more expensive

resolution def

ability to distinguish two closely related and/or eluting chromatographic peaks

bigger diameter effects

dec efficiency, inc loadng capacity, inc flow rate, inc analysis time

thicker column effects

longer retention time, lower resolution, broader peak, good capacity

shark fin peak

capacity overloaded, fronting, shape distorted

tailing peak

asymmetrical, from active site at injection or column

gc maintenance 

change liner, wool, septum, and column or clip 

gcms contamination source

fingerprints, air leak, cleaning solvents, column bleed, dirty materials

Retention time locking 

use known molecule with known rt on GC, the instrument will adjust pressure to return molecule to the correct time

isomers differentiation 

fragment the same, only differentiate through retention time

fix column bleed

install new column when background noise gets really high

why choose derivatization

Enhance detectability, enhance excitability, stabilize components, increase retention, reduce retention, improve sensitivity, improve gc/ms appearance, bulk weight

which step to derivatize

can be performed at any steps

flash derivatization

occurs very last minute, within injection port

Mass spec instrument components

sample introduction, transfer region, high vacuum, pumps, ionization source, analyzer, detector, data processor

GCMS diagram

see photo

electron ionization

disrupt electron cloud to cause loss of an electron and create a radical cation