Chromatography And Analysis Flashcards
Summary of Phase I Metabolism
- Almost any chemical transformation can be catalyzed by enzyme systems, mainly in the liver
- These systems developed primarily to process endogenous compounds and dietary xenobiotics
- Many xenobiotics are substrates for a number of different Phase I reactions, e.g. diazepam
- Phase I reactions can be used to activate or alter the activity of drugs, but are primarily employed to prepare xenobiotics for Phase II processes
Cytochrome P450 Oxidations
O-dealkylation
Codeine –> (CYP2D6) Morphine
H3CO –> HO
Summary of Phase II Metabolism
- Reactions are generally with Phase I products
- Common requirement for an energy rich or “activated” intermediate
- Products are generally more water soluble and are ready for excretion
- There are many complementary, sequential and competing pathways
- Together with phase I metabolism, this is a coupled interactive system interfacing with endogenous metabolic pathways
Glucuronidation
ROH - not excreted in large amounts
UDP-glucuronosyl transferase - not water soluble
Glucuronide conjugate (B) - water soluble, excreted in large quantities
Glucuronide will be hydrolysed by enzymes
by increasing stationary phase
retention can be increased
Chromatography
- A technique used to analyse and separate a mixture of compounds into individual components
- Stationary phase versus mobile phase
- Traditional view is that separation is achieved by the distribution of molecules between a stationary phase and a mobile phase
- Many different chromatography experiments
- Chromatography can be linked with other methods of analysis (hyphenated techniques)
Chromatographies
- Adsorption chromatography, e.g. TLC, column
- Stationary phase is solid, e.g. silica, alumina
- Partition chromatography, e.g. HPLC
- Both stationary and mobile phase are ‘liquids’
- Ion-exchange chromatography
- Stationary phase is an ion-exchange resin
- Gel permeation chromatography
- Size-exclusion chromatography
- Affinity chromatography
- Ligand immobilised on solid, stationary phase
Thin Layer Chromatography (TLC)
qualitative technique
Rf of compound 1 = X1/Xs
• Silica stationary phase: non-polar compounds eluted first
• Non-polar versus polar solvents, reverse phase TLC
• Visualisation using UV, iodine, sulfuric acid, molybdate
what phases used mostly in TLC
polar mobile phases
‘Lab-Scale’ Column Chromatography
- qualitative technique
• Sometimes viewed as a black art
• Typically the separation of organic compounds on an inert stationary phase, e.g. silica or alumina
• Column packing method can affect results
• Gravity columns driven by the solvent head
• Flash column chromatography is driven by the application of pressure to the solvent head
• Must do TLC first before starting column and carry out TLC throughout to detect eluted compounds
• Excellent technique but preparative, not analytical
Analytical Techniques
- Used for the quantitative analysis of components of a (complex) mixture e.g. a pharmaceutical formulation
- Analytes are typically separated based on differing affinities for the stationary and mobile phases
- Mobile phase can be a gas e.g. gas chromatography (GC) or a liquid e.g. high performance liquid chromatography (HPLC)
- HPLC is widely used in pharmaceutical analysis
Retention factor k
- measures how long the material is retained in the column
- retention factor is affected by the mobile stationary phase
• Independent of column length or flow rate
• Need to measure column dead time to - using dead time you can get your retention factor
Separation Factor a
- Identifies when peaks elute relative to one another
- Ratio of the retention factors (k2 > k1)
- Separation factor >1 to achieve separation
- Governed primarily by the stationary phase selection
a =
k2 / k1 = tr,2 - to/ tr,1 - to
Column Efficiency (Plate Number) N
• Represents narrowness of the peak
• Columns with large values of N give narrower peaks
-the narrower the peak, the better the separation
-the larger the number of plates you have, the higher the amount of separation
-the greater the depth of stationary phase , the better the separation
-the more densely the column in HPLC is packed, the greater the efficiency
what is plate number and equation
each layer is called a plate
N = 5.54 (tr/w0.5)2
Asymmetry Factor As
- In practice, peak shapes are not gaussian and have ‘tails’
* As 0.9 → 1.2 acceptable; As >1 tailing, As <1 fronting
When does tailing increase
tailing increases when the column becomes worn out, the more tailing you get the more likely the peaks are to overlap
Resolution R
- Ideal valley between peaks should return to the baseline
- R is a quantitative measure of separation
R = (square root)N / 4 x k/k + 1 x a-1/a
don’t need to remember the formulae, just understand it
• To achieve resolution:
- Peaks should be retained on the column (k > 0) - need to have a decent retention time
- Peaks have to be separated from one another ( > 1) - need a good value of alpha
- The column must develop some min value of N - the more efficient the column, the greater the resolution. the older the column, the older the mobile phase
Varying Conditions
resolution affected by the efficiency of the column and mobile phase
- initial
- vary k’ - if retention time is increased
- increase N - brand new column, more narrow peaks
- increase a - combination of a good efficient column and mobile phase
Gas Chromatography (GC)
- turns material into gas
-used to detect volatile agents but not small molecules
• Sample is injected on to column (i.d. 0.1–0.5 mm 60 m)
• The column is heated to release the volatile components
• The mixture is separated on the column and various methods used to detect each component
High Performance Liquid Chromatography (HPLC)
- The sample is injected as a solution on to the column
* The mixture is separated on the column and each of the components is detected using some means
HPLC Overview
- A typical apparatus consists of a mobile phase reservoir, high pressure pump, injection valve, sample loop, column and detector
- Mobile phase flows constantly through the pump, column and detector
- Injection valve introduces the sample to this flow
HPLC Columns
- Stainless steel components, able to withstand high pressures and wide range of solvent systems
- Commonly 10 cm in length with an internal diameter (ID) of 4.6 mm (can be 5 – 25 cm in length)
- Stationary phase is packed into the stainless steel column under high pressure
- Stationary phase commonly consists of silica particles with a diameter of 5 μm but can be as little as 1.7 μm
- Stationary phase can be modified to allow separation of different molecules e.g. chiral stationary phase used to separate enantiomers
Normal vs Reverse Phase HPLC
- Historically the stationary phase consisted of unmodified silica (Si-OH) and was more polar than the mobile phase (normal phase HPLC)
- In >95% of modern HPLC analysis the silica has been modified to make it less polar (reverse phase HPLC)
- Typical modifications include the addition of C18 alkyl groups to the silica surface (octadecylsilyl, ODS)
- The choice of column depends on the analytes to be separated e.g. analytes that are non-polar will be retained for longer on a reverse phase column and vice-versa – RP-HPLC used most frequently
Reverse phase HPLC and Log P
- Log P is a measurement of the hydrophobicity of a particular molecule
- Analyte retention is based on the interaction between the analyte and the mobile and stationary phases
- Interactions with the mobile phase become important
Reverse phase HPLC and higher Log P
• The higher the Log P of a molecule the more it interacts with the ODS stationary phase, therefore it’s retention time is increased
Reverse phase HPLC and lower Log P
• The lower the Log P of a molecule the less it interacts with the ODS stationary phase, therefore it’s retention time is decreased
Reverse phase (RP) HPLC and pH - What is the phosphoric acid for?
adjusts the pH of the mobile phase
RP-HPLC and pH
• A significant number of pharmaceutically relevant molecules contain ionisable functional groups
• Poor retention/peak shape is often observed for ionised analytes
• A molecule being analysed should be predominantly in a non-ionised form
• For acids, adjusting the pH of the mobile phase to 1 pH unit below the pKa of the analyte ensures 90 % of the analyte is in the non-ionised form (1 pH unit above the pKa for bases)
-pH affects retention times
Material for RP-HPLC
material needs to be non-ionised to be analysed by RP-HPLC
Effect of pH on the retention of a basic analyte during reverse phase HPLC
- Retention factor is reduced at low pH
* Retention factor is increased at high pH
Effect of pH on the retention of an acidic analyte during reverse phase HPLC
• Retention factor is reduced at high pH
• Retention factor is increased at low pH
- for acids need to have a low pH
Detectors
• Range of detectors available for both GC and HPLC
- Most based upon spectroscopic or colorimetric method
- Mass spectrometry
- Flame ionisation
- UV/vis detector: flow-through cell
- Diode array detector: flow-through cell
- Fluorescence detector: flow-through cell
- Apparatus linked to some other means of analysis
HPLC in pharmaceutical analysis
-Widely used in all aspects of pharmaceutical dosage form development and manufacture including:
HPLC in pharmaceutical analysis
• Drug discovery
• Pre-formulation
• Impurity testing of API and excipients
• Formulation
• Quality assurance (QA) testing e.g. API content, stability studies, impurities, degradation products
• Pharmacokinetic and drug metabolism studies
• Separation and quantification of chiral API isomers
‘Hyphenated’ Techniques
- GC-MS: GC coupled to MS
- LC-MS: HPLC coupled to MS
- MS-MS: tandem mass spectrometry
LC-MS
• The coupling of HPLC with mass spectrometry (MS)
- observes ions, doesn’t detect neutral species
• More difficult than coupling GC to MS since the analyte is already in an inert gaseous phase
• Mobile phase must be removed before MS
• The analyte(s) must be ionised prior to entering the mass spectrometer
• A range of ionisation methods e.g. ESI, EI, APCI
• Used when the identity of the analyte is unknown or it shows poor intensity using other detectors
LC-MS diagram
Inlet (solid sample chromatography) -> Ion source ( EI, CI, FAB, MALDI, ESI, APCI) -> Mass filter (Magnetic sector, quadrupole, time of flight, ion trap) -> Detector -> Spectrum
- HPLC flow rate typically 1 mL/min or less
- Polarity of the ionisation source (+ve or –ve) depending on analyte
- Detector produces a signal proportional to the number of ions impacting it
Applications of LC-MS in Pharmaceutical Analysis
- Mainly used where the analyte(s) are unknown and require identification
- Drug discovery (characterisation)
- Drug metabolism studies (in vitro and in vivo)
- Analysis and identification of impurities e.g. degradation products in pharmaceutical formulations
- Determination of active/inactive chiral impurities in API e.g. stereoisomers
The Mass Spectrometer
- Almost modular, dependent upon intended analyte
- Sensitive
- Destructive
- Requires high vacuum
- Automated in hyphenated techniques
Inlet (Volatile or Solid Sample,Chromatography effluent) -> Ion Source (Ionisation techniques EI, CI, FAB - MALDI, ESI, APCI) -> Mass Filter (Magnetic Sector Quadrupole (quad) Time Of Flight (TOF) Ion Trap FT-ICR) -> Mass Filter (MS-MS MS-MS-MS) Detector -> analysis
Ionisation Techniques
- ‘Hard’ techniques
• Electron Ionisation (EI) (much fragmentation) - ‘Soft’ techniques (more likely to observe molecular ion)
• Chemical Ionisation (CI) (fragmentation)
• Fast Atom Bombardment (FAB)
• Electrospray Ionisation (ESI) (no vacuum)
• Matrix-Assisted Laser Desorption Ionisation (MALDI)
• Atmospheric Pressure Chemical Ionisation (APCI)
Mass ‘Filtration’/Analysis
Time Of Flight (TOF)
• Electrostatic sector + magnetic sector = HRMS
• Quadrupole (Quad)
Interpretation: Molecular Ion
- In a clean sample, i.e. one compound, the molecular ion is the radical cation formed from the compound of interest
- For the majority of techniques it is the highest observable ion but may not be the most intense peak
- The peak with maximum intensity, i.e. 100%, is termed the base peak and intensities are measured relative to it
- Possible to estimate molecular formula if no CHN%
- The Nitrogen Rule
- Even m/z molecular ion = even number of N (or zero)
- Odd m/z molecular ion = odd number of N
Interpretation: Fragmentation
- Fragmentation is essentially the molecule falling apart/ disintegrating inside the mass spectrometer
• Ionisation techniques result in more/less fragmentation
• Collisions between molecular ions and fragment ions
• Fragmentation and rearrangement can occur
• Loss of neutral molecules to give stable ions - Valuable structural information; fragments and losses
- Fingerprint: like IR, each molecule has a unique mass spectrum when measured under identical conditions
- It is not necessary to identify every ion in a spectrum but knowing a few significant patterns and ions is useful
Interpretation: Fragmentation
One-bond cleavage
Mol• (radical) ecule+ (Even-electron ion, ‘Low’ energy, Few further fragments)
Interpretation: Fragmentation
Two-bond cleavage
Molec (neutral) ule•+ (Odd-electron ion, Still high energy, Further fragments likely)
mass spec only shows
mass spec only shows radicals, not neurtal
Metabolism of Benzodiazepines
Diazepam
Diazepam –> (CYP3A4) Temazepam –> (CYP2C19) R-Oxazepam –> (UGT21A9, UGT2B7) Glucuronidation
Diazepam –> (CYP2C19) Nordazepam –> (CYP3A4) S-Oxazepam –> (UGT2B15) Glucuronidation
Metabolism of Benzodiazepines
Alprazolam
Alprazolam –> (CY3A5, CY3A4) Hydroxylation –> Glucuronidation
Metabolism of Benzodiazepines
Triazolam
Triazolam –> (CY3A5, CY3A4) Hydroxylation –> Glucuronidation
Metabolism of Benzodiazepines
midazolam
midazolam –> (CY3A4) Hydroxylation –> (UGT1A4, UGT2B7, UGT2B4) Glucuronidation
midazolam –> (UGT1A4) Glucuronidation
Metabolism of Benzodiazepines
flurazepam
flurazepam –> Hydroxylation/Alkylation –> Glucuronidation
Metabolism of Benzodiazepines
bromazepam
Bromazepam –> (CYP2D6, CYP1A2) –> Hydroxylation –> Glucuronidation
Metabolism of Benzodiazepines
lorazepam
lorazepam –> (UGT2B15) Glucuronidation
Metabolism of Benzodiazepines
clonazepam
clonazepam –> (NAT2) Acetylation –> Elimination
