Controlled Substances

Partitioning

occurs at phase boundary due to a greater affinity for one phase than the other

• liquid-liquid (water and oil)

• solid-liquid (ice in water)

• liquid-gas (boiling water)

physical states: solid, liquid, gas

function of temp and pressure → water is classic example

based on chemical properties

• charged vs neutral

• polar vs non-polar (polarity: separation of charge- dipole moment)

• density, pH, etc

Important for chromatography as well

Equilibrium

the state where the concentration of all reactants and products remain constant with time

• rate of forward reaction is equal to the reverse reaction

• equilibrium constant (K)

• express the relationship between the products and reactants

• [A] = concentration of chemical species A

• ^a = stoicheometric coefficient from the balanced equilbirum equation

Le Châtelier’s Principle

disturbance of equlibrium results in a counteraction to re-establish eqilibrium

• K = equilibrium constant

  • K ~ 0.001, mostly reactants

  • K ~1000, mostly products

  • only calculated when reaction is at equilibrium

reaction moves to left or right to reach equilibrium again

Henry’s Law

related the partial pressure of a gas to the concentration in a liquid

• ex) ethanol is more volatile than the matrix components from blood

Breathalyzer

partition between liquid and gas in the lungs

Acid Dissociation Constant (Ka)

quantitative measure of the strength of an acid

• equilibrium constant of the dissociation of a weak acid in an aqueous solution

• strong acids completely dissociate (ionize)

  • HCl → H+ + Cl-

  • HNO3 → H+ + NO3-

• often reported as pKa due to the many orders of magnitude

Solubility Equilibrium Constant (Ksp)

solubility of a compound in an aq phase

• salt vs. freebase form

• common salts: sodium, calcium, sulfates, and chlorides

• don’t forget about pH

Characterization of Solubility

• hydrophilic/lipophobic = soluble in aq phase

• hydrophobic/lipophilic = non-soluble in aq phase

Salt Form Base

• charged species

• counter ion

• soluble in aq phase (water)

• ex) cocaine hydrochloride (snorted/injected)

Freebase Form

• uncharged species

• no counter ion

• non-soluble in an aq phase (water)

• ex) freebase cocaine (smoked)

Lipophilicity

solubility of a drug in lipid membranes

• hydrophobic = lipophilic

why does it matter?

• our biological fluids are aq

• our membranes are lipids

• drug absorption through passive diffusion

metabolic properties → drug metabolism

• ADME = Absorption, Distribution, Metabolism, and Excretion

Pharmacokinetics

movement of drugs in the body

Pharmacodynamics

effect of drugs and mechanism of action

Octanol-Water Partition Coefficent (Kow)

how lipophilicity is calculated

• partition coefficient of the unionized form

• + logP = lipophilic

  • ex) Isopentanol → logP ~ 1 (10:1 ratio of octanol:water)

• - logP = hydrophilic

  • ex) Methanol logP ~0.7 (1:7 ratio of octanol:water)

• Equal partitioning when logP = 0

• find where something wants to be (octanol or water) by shaking everything up and see where the molecule wants to be (aq or organic) → good to know for extraction and sample prep

Distribution Coefficient (LogD)

represents the partition coefficient for both the neutral and ionized form of the compound at any pH

• logD of 7.4 = real physiological and biological conditions

• determine the pH → this one wants to stay in blood

  • to get rid of it → go somewhere in the body that has a higher pH

  • helpful to know what drug— helps determine where it wants to be in the body

Bronsted-Lowry Definitions

Acid = donates a proton (conjugate base)

Base = accepts a proton (conjugate acid)

Lewis Definitions

Acid = accepts a pair of valence electrons to form a bond

Base = donates a pair of valence electrons to form a bond

Strength of an acid

tendency to donate a proton and/or dissociate in water

• ex) HCl → H+ + Cl-

pH

measurement scale to specify the acidity or basicity of an aqueous solution

• logarithmic scale = each unit is a 10-fold change in acidity or bascitiy

• range from 0-14 (7 is neutral)

• really a measure of the amount of free H+ and OH- ions in aq soln

Kw

self-ionization constant of water = 14

pKa

another measure of acid strength

• converts large Ka values into manageable numbers

• smaller pKa = stronger acid

Henderson-Hasselbalch

provides the relationship between pH and pKa

• acids = pH and pKa

• bases = pOH and pKb

Amphoteric

more than one ionizable center

• acidic and basic character

• ex) morphine

Does partitioning require both liquids to be immiscible or not?

IMMISCIBLE

What are the 5 extraction techniques?

1. Dry Extraction

2. Liquid-Liquid Extraction

3. Liquid-Liquid Microextraction

4. Solid-Phase Extraction

5. Solid-Phase Microextraction

Dry Extraction

most simplistic extraction

• add an organic solvent to a powdered sample

• extraction of drugs from common diluents like sugar and cornstarch

• unfortunately, often too simple an extraction —> if several things, can’t just put this extraction in IR unless combined with chromatographic separation

• ex) GC-MS of unknown white powder (how many things are there?)

Liquid-Liquid Extraction (LLE) aka Solvent Extraction

• aqueous and organic solvents separated via separatory funnel (shaking forces interaction)

• partitioning driven by pH dependence

• solvent must be immiscible to form 2 layers

• density is important → which layer do I want?

Advantages:

• quick & easy for qualitative analysis

• high selectivity

• high eluant capacity (can do with large samples/bulk quantity)

Disadvantages:

• large solvent consumption

  • cost of solvent

  • cost for disposal

  • safety concerns → benzene & chloroform

• emulsions possible = ruin the experiment (might not be able to test the samples again)

• bulky and expensive glassware

H2O density

1.000 g/mL

Liquid-Liquid Microextraction

same principles as LLC, but on a smaller scale

• solvent volumes less than 5 mL

• centrifuge tubes instead of a separatory funnel

• must carefully remove the desired layer with a pasteur pipette

Advantages: lower solvent consumption & associated concerns

Disadvantages: difficulty removing layers (have to be extremely careful)

Solid-Phase Extraction (SPE)

better for quantitative analysis

• dissolved analytes separated from the solution

• affinity of solute for sorbent material relative to solution

• eluted from sorbent material

• different stationary phases available

• positive or negative pressure used to force/pull the sample through the cartridge

  • let gravity do its thing OR do it faster with pressure

Solid-Phase Partitioning

• solutes dissolved in a mobile phase and pass through a stationary phase

• separation occurs based on interaction (adsorpton, solubility, binding, or electrostatic interactions)

• K_D is used to express degree of interaction

  • KD = ([stationary]/[mobile])

• polarity-based interactions (like-dissolves-like)

• same concept as liquid chromatography

• now getting to a point to separate each component individually without having crossover (more selective technique)

• based on interaction desired → normal phase, reversed phase, ion exchange

Advantages:

• enhanced separation compared to LLE → upwards 50x better than LLE

• greater recovery → no transfer losses between glassware

• reduced organic solvent waster → minimal organic solvent required

• less apparatus required → no expensive and bulky separatory funnels

Disadvantages:

• more complex chem involved → wide range of sorbent material and elution solvents

• requires method development → more time-consuming on the front end

• more steps in the extraction process → conditioning, washing, elution, etc.

• cost of SPE cartridges and pressure system

Normal Phase

polar bonded phase / nonpolar solvent

• retains polar compounds → if separating polar things, do this

• elute with polar solvent

Reversed Phase

non-polar bonded phase / polar solvent

• retains non-polar compounds

• elute with non-polar solvent

Ion Exchange

anion (positively charged) → retains negatively charged compounds

cation (negatively charged) → retain positively charged compounds

SPE Process

1. Conditioning the sorbent material → wets the surface

2. Load the sample solution → allows for interaction with the sorbent material

3. Wash the sorbent material → removes remaining impurities

4. Elute analytes from sorbent material → removes analytes from the sorbent material and into the final analyte solution

**same holds true for polarity or ionic types

• elute in increasing strength (weakest to strongest)

SPE Concerns

• binding capacity and surface area determine the volume of soln

• based on saturation of binding sites

• once all binding sites are filled, no more analytes can be retained

• leads to loss of analytes

• sample can be concentrated with minimum solvent or through dry down and reconstitution (adding expenses)

Solid-Phase Microextraction (SPME)

fiber coated with the extracting phase → PDMS with an additive is common

• liquid or gas sample (solvent free method)

• targets volatile & semi-volatile compounds

• sample heated and allowed to reach equilibrium between the headspace and solution

• small fiber acts as the substrate for the sorbent

  • active material bonded to a thin silica needle

  • liquid polymer or solid sorbent

  • similar materials to SPE

• extraction and preconcentration

• equilibrium is important for quantification

• common for ILR analysis with SPME-GC-MS

Advantages:

• target for volatile and semi-volatile compounds

• preconcentration step

• automation

Disadvantages:

• lower effectiveness due to reduced surface area of the SPME fiber

• requires method optimization for fiber selection and equilibration period

• only limited commercially available SPME chemistries