Lec 20 - Sample Preparation in the Tox Laboratory

what are sample matrices

everything but the analyte of interest (i.e. stuff in urine excluding the drug being measured)

problems caused by sample matrices when measuring analytes

• matrix effects

• reduced sensitivity

• unreliable quantitation during analysis --> therefore we need to do sample prep!

reasons sample prep optimizes sample for analysis

• removes or extract drug/analyte from biological matrix
• isolates it from endogenous constituents that may interfere with analysis
• concentrates drug/analyte
• can dissolve drug in a solvent appropriate for anaytical method (i.e. volatile for GC or dissolved in aqueous solution for LC, IA, spec)

sample prep techniques

1. dilute and shoot
2. homogenization
3. protein precipitation
4. liquid-liquid extraction (LLE)
5. solid-phase extraction (SPE)
6. hydrolysis
7. derivitization

dilute and shoot

liquid specimen is diluted in a solvent and internal standard is added

when would you use dilute and shoot

for LC-based techniques, in situations where we do not need increased sensitivity

ways to trap constituents from matrix when using dilute and shoot

• centrifugation
• filtration
• guard columns

advantages of dilute and shoot

fast, cheap, easy

disadvantages of dilute and shoot

• additional costs with consumables
• increased routine and preventative instrument maintenance
• reduced analytical sensitivity
• doesn't rlly remove matrix components and interferences

risks when diluting a sample

too diluted: not detectable not diluted enough: saturate instrument

homogenization

for solid biological matrices where a device creates a uniform mixture

homogenization devices

rotor-stator, ultrasonic/sonicator, bead mill, ball mill

rotor-stator

fast blade shears soft tissues (liver, kidney, brain) like a blender

ultrasonic/sonicator

high-frequency sound waves lyse soft tissues

bead mill

sample is disrupted by intense shearing and collision with beads

ball mill

grinding due to impact as container roates and pulverizes sample (usually hair) to a powder

advantages of homogenization

• improve sample uniformity/reduce variability
• disrupt tissues and cells to improve analyte accessibility
• compatible with multiple extraction techniques
• applicable to various sample types

disadvantages of homogenization

• heat-sensitive analytes may be degraded
• increased matrix interference by liberating constituents like proteins or lipids
• risk of cross-contamination
• additional clean-up required
• time consuming

protein precipitation

removal of proteins from biological samples to help liberate protein bound analytes and eliminate interferences in downstream analysis

protein precipitation steps

precipitation, then separation step by centrifugation or filtration, then add internal standard

preferred solvent for protein precipitation

organic solvents

advantages of protein precipitation

• easy
• minimal loss
• inexpensive reagents
• not labour intensive
• ease of automation

disadvantages of protein precipitation

• limited selectivity
• incomplete removal of proteins
• matrix effects can suppress ionization and reduce sensitivity
• loss of analyte due to co-precipitation

liquid-liquid extraction

separates analytes based on difference in solubility b/w 2 immiscible liquid phases (usually aqueous and organic)

purpose of LLE

isolate and potentially concentrate analytes from a complex matrix by transferring to a solvent so it is more soluble and easier to analyze

what partitions in organic phase

uncharged, less polar --> nonpolar solvent

what partitions in aqueous phase

charged, more polar --> polar solvent

what happens when pH < pKA

acidic unionized basic ionized

what happens when pH > pKA

acid ionized basic unionized

amphetamine

basic drug: weak base w/ primary amine, pKA = 9.8

simple extraction method for basic drugs

specimen + pH 12 buffer + solvent (i.e. chloroform) --> mixed + centrifuge --> do GC/LC on organic layer, aqueous layer = waste

which layer would amphetamine settle in when doing simple extraction

organic layer (bottom layer)

back extraction

more specific method of extraction, gets rid of more interferences than doing a simple would

back extraction process

same as simple extraction but you would add acid to organic layer to make basic drug ionized, then add base again and extract as normal --> neutrals (fats) and acidic drugs are left behind

extracting drugs with 2+ ionizable functional groups

functional group with smaller pKA dissociates first like a rate limiting step

what do we do for amphoteric drugs

we have both acidic and basic functional groups so solid phase extraction might be better

advantages of LLE

• simple
• good repeatability
• minimal equipment required

disadvantages of LLE

• labour intensive
• large volumes of hazardous solvents
• not all drugs are stable at extreme pH
• strong acids/bases can cause bubbles (emulsification)
• other contaminants may be extracted and interfere with analysis

solid phase extration

analytes in liquid mixture are partitioned between a liquid and solid (stationary phase)

separation principal behind SPE

takes advantage of physical and chemical properties of drug that dictate adsorption, solubility, binding, and electrostatic interactions

modes of SPE

• reverse phase
• normal phase
• ion-exchange
• affinity

steps in SPE

1. conditioning w/ organic solvent (i.e. methanol) or water, makes sure surface is ready and wet enough or it will crack
2. sample is loaded
3. washing step to remove contaminants, then column is dried with air or nitrogen
4. use solvent to elute metabolite of interest

advantages of SPE

• more separation power
• selective
• safe--no exposure to harsh chemicals
• no emulsion problems
• easily automated
• efficient - less time, effort, and solvent vs LLE

disadvantages

• pre-treatment required for viscous samples
• sorbent clogging
• can have poor reproducibility b/w different sorbent lots
• possibility of low recovery

hydrolysis

can be enzymatic or chemical to remove bound drugs and metabolites that may be interfering with analysis

glucuronidation

phase 2 metabolism reaction drugs undergo

what does glucuronidation do to drugs

it is rlly polar so it can reduce drug activity when conjugated and make them more water soluble so they are excreted easier

problems with glucuronide metabolites during analysis

• not typically analyzed by GC
• reverse-phase LC doesn't retain metabolites well, reducing quantitative accuracy

what conditions are required for enzymatic hydrolysis

optimal time, temperature, pH, and concentration

enzyme hydrolysis advantages

• improve sensitivity
• widely applicable
• mild temperature and pH conditions

enzyme hydrolysis disadvantages

• incomplete hydrolysis and not universal
• long incubation time for some enzymes
• enzyme variability
• T/pH sensitivity

chemical hydrolysis

removal of conjugated groups under acidic or alkali conditions

advantages of chemical hydrolysis

• low cost
• wide range of conjugates
• predictable and reproducible

disadvantages of chemical hydrolysis

• harsh conditions--high temperature and strong acids/bases (may not work for temperature labile analytes)
• safety concerns
• extra sample cleanup required

chemical derivatization

enhance volatility, temperature stability and detectability of drugs; super important for testing analytes with polar groups using GC/MS

principal behind derivatization

replacement of active hydrogen on various functional gorup

general reaction for derivatization

R1-AH + R2-D -> R1-AD + R2-H A = O, N, S H = hydrogen D - functional group on derivatizing agent

common derivatization techniques

silylation, acylation, alkylation

silylation

• most universal derivatization technique
• works for alcohols, phenols, amines, thiols, and -COOH
• introduces silylation group through substitution at active hydrogen

examples of silylation reagents

MSTFA and BSTFA

acylation

introduction of acyl group (RCO-) onto molecule through substitution of active hydrogen; good for polar molecules with hydroxyls, thiols, or amines -> esters, thioesters, amides

acylation reagents

TFAA, PFPA or PFAA, HFBA, HFAA

can you do acylation for carboxylic acids

no, do silylation instead

alkylation

• not as common
• sub alkyl or aryl group at an active hydrogen on hydroxyls
• mainly done for modificiaation of acidic hydrogens on COOH and phenols
• can be used in conjunction with silylation and acylation

common reagent for alkylation

BF3/methanol and PFBBr

advantages of derivatization

• decreased polarity
• increased molecular mass
• improved detectability
• increased specificity
• enhanced volatility
• increased temperature stability

disadvantages of derivatization

• increased sample prep time
• clean-up for excess agent or by-products
• if u don't clean up can mess up stationary phase, detector fouling, and increased instrument maintenance