Chromatography And Analysis Flashcards
1
Q
Summary of Phase I Metabolism
A
- 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
2
Q
Cytochrome P450 Oxidations
A
O-dealkylation
Codeine –> (CYP2D6) Morphine
H3CO –> HO
3
Q
Summary of Phase II Metabolism
A
- 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
4
Q
Glucuronidation
A
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
5
Q
by increasing stationary phase
A
retention can be increased
6
Q
Chromatography
A
- 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)
7
Q
Chromatographies
A
- 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
8
Q
Thin Layer Chromatography (TLC)
A
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
9
Q
what phases used mostly in TLC
A
polar mobile phases
10
Q
‘Lab-Scale’ Column Chromatography
A
- 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
11
Q
Analytical Techniques
A
- 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
12
Q
Retention factor k
A
- 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
13
Q
Separation Factor a
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
14
Q
a =
A
k2 / k1 = tr,2 - to/ tr,1 - to
15
Q
Column Efficiency (Plate Number) N
A
• 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
16
Q
what is plate number and equation
A
each layer is called a plate
N = 5.54 (tr/w0.5)2
17
Q
Asymmetry Factor As
A
- In practice, peak shapes are not gaussian and have ‘tails’
* As 0.9 → 1.2 acceptable; As >1 tailing, As <1 fronting
18
Q
When does tailing increase
A
tailing increases when the column becomes worn out, the more tailing you get the more likely the peaks are to overlap
19
Q
Resolution R
A
- 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
20
Q
• To achieve resolution:
A
- 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
21
Q
Varying Conditions
A
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
22
Q
Gas Chromatography (GC)
A
- 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
23
Q
High Performance Liquid Chromatography (HPLC)
A
- 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
24
Q
HPLC Overview
A
- 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
25
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
26
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
27
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
28
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
29
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
30
Reverse phase (RP) HPLC and pH - What is the phosphoric acid for?
adjusts the pH of the mobile phase
31
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
32
Material for RP-HPLC
material needs to be non-ionised to be analysed by RP-HPLC
33
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
34
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
35
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
36
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
37
‘Hyphenated’ Techniques
* GC-MS: GC coupled to MS
* LC-MS: HPLC coupled to MS
* MS-MS: tandem mass spectrometry
38
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
39
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
40
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
41
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
42
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)
43
Mass ‘Filtration’/Analysis
Time Of Flight (TOF)
• Electrostatic sector + magnetic sector = HRMS
• Quadrupole (Quad)
44
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
45
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
46
Interpretation: Fragmentation
One-bond cleavage
| Mol• (radical) ecule+ (Even-electron ion, ‘Low’ energy, Few further fragments)
47
Interpretation: Fragmentation
Two-bond cleavage
| Molec (neutral) ule•+ (Odd-electron ion, Still high energy, Further fragments likely)
48
mass spec only shows
mass spec only shows radicals, not neurtal
49
Metabolism of Benzodiazepines
| Diazepam
Diazepam --> (CYP3A4) Temazepam --> (CYP2C19) R-Oxazepam --> (UGT21A9, UGT2B7) Glucuronidation
Diazepam --> (CYP2C19) Nordazepam --> (CYP3A4) S-Oxazepam --> (UGT2B15) Glucuronidation
50
Metabolism of Benzodiazepines
| Alprazolam
Alprazolam --> (CY3A5, CY3A4) Hydroxylation --> Glucuronidation
51
Metabolism of Benzodiazepines
| Triazolam
Triazolam --> (CY3A5, CY3A4) Hydroxylation --> Glucuronidation
52
Metabolism of Benzodiazepines
| midazolam
midazolam --> (CY3A4) Hydroxylation --> (UGT1A4, UGT2B7, UGT2B4) Glucuronidation
midazolam --> (UGT1A4) Glucuronidation
53
Metabolism of Benzodiazepines
| flurazepam
flurazepam --> Hydroxylation/Alkylation --> Glucuronidation
54
Metabolism of Benzodiazepines
| bromazepam
Bromazepam --> (CYP2D6, CYP1A2) --> Hydroxylation --> Glucuronidation
55
Metabolism of Benzodiazepines
| lorazepam
lorazepam --> (UGT2B15) Glucuronidation
56
Metabolism of Benzodiazepines
| clonazepam
clonazepam --> (NAT2) Acetylation --> Elimination
