Lec 2- HPLC

Question 1

Analysis of drugs by HPLC

Answer 1

• Solvents => Pump => Injector => Column => Detector => Recorder
• Can be considered as a modular system
• How many stationary phases (Based on the mechanism of separation) are there in LC?
• Which is most commonly used?

Question 2

Using reverse phase HPLC

Parameter changed= Column length

Answer 2

1. Increased column length

• Results in Increased direction of change
• And Increased retention time
• Increased Resolution ​

2. Decreased Column length

• Decreased direction of change
• Decreased retention time
• Decreased resolution

Question 3

Using reverse phase HPLC

Parameter changed= Flow rate

Answer 3

1. Increased flow rate

• Increased direction of change
• Similar retention time- often decreased retention time
• Decreased resolution

2. Decreased flow rate
   * Decreased direction of change
   * Similar retention time- Often increased retention time
   * Increased resolution

NB- The degree of separation is most easily controlled by changing a mobile phase composition

Question 4

Determination of methotrexate in plasma by HPLC

Answer 4

• What diseases do the first 2 drugs treat

Question 5

1. Clean up procedures

Q1) Where was the other 60% of the MTX?

Q2) Was this likely to cause errors?

Q3) How can these errors be compensated?

Answer 5

• Protein was precipitated by adding 0.3 ml of 10% trichloroacetic acid to every 1 ml of plasma
• Following centrifugation, MTX could be measured with a UV detector at 305nm
• Recovery of MTX in the acid extracts was reproducible at about 40% (38 to 41% in 9 determinations)

Q1) Where was the other 60% of the MTX?

• It is likely that it was bound to the precipitated protein

Q2) Was this likely to cause errors? - Yes

Q3) How can these errors be compensated? By adding internal standard

Question 6

2. Derivatisation

Q1) Why were the drug and IS oxidised

Answer 6

• For each 0.5ml of trichloracetic acid extract, 0.05ml of 5M acetic acid- Na acetate buffer (pH 5.0) was added
• Oxidation was performed at room temperature in 5 minutes by the addition of 5% KMnO₄ (aq) solution
• The samples were decolourized by the addition of 0.1 ml of a 3% solution of H₂O₂
• This procedure resulted in the quantitative conversion of MTX to 2,4-diaminopteridine-6-carboxylic acid
• When 2-hydroxyfolic acid was used as the internal standard it was added to the trichloroacetic acid solution to give a final concentration equivalent to 0.5ug/mL in plasma
  
  • To convert them into fluorescent derivatives to improve the detection sensitivity

Question 7

3. Analysis

• Q1) What was the stationary phase?
• Q2) Was it reverse phase or normal phase HPLC
• Q3) Tris?

Answer 7

• Water 6000 pumps were used with a uBondpack C₁₈ column (4mm x 30cm) and a model U₆K injector
• A SF-770 multiple wavelength UV detector and model FS (fluorescence detector) 970 detector were connected in series to the outlet of the column
• 2 solvent systems were used
• Q1) C-18
• Q2) A reverse phase
• Q3) Tris(hydroxymethyl) aminomethane: (CH₂OH)₃CNH₂

Question 8

Answer 8

NB look at BB

Question 9

4. HPLC trace

Answer 9

• The HPLC traces for MTX analysis by HPLC and fluorescence detection
• Samples of human plasma were ‘spiked’ with MTX (0.1 ug/ml) and 2-hydroxyfolic acid (0.5 ug/ml)
• Samples (100 ul) of oxidised plasma extracts were loaded onto the chromatograph A
• Plasma plus MTX and the internal standard; B

Question 10

5. Calibration curve

Answer 10

• Plasma containing 2-hydroxyfolic acid (0.5 ug/ml) and different levels of MTX was extracted, derivatized and analysed as discussed (6 samples, each injected 3 times )
• MTX plasma standard curve with the use of an internal standard

Question 11

Assay procedure of paracetamol tablets using a calibrate curve

Answer 11

1. Weigh out 125mg of paracetamol standard and dissolved in 250ml of 0.05M aq. acetic acid (stock solution)
2. Prepare a series of standard solution each containing 0.5, 1.0, 1.5 & 2.5mg/ 100ml of the drug from the stock solution
3. Weigh out powder 20 tablets (total 12.1891 g)
4. Weigh out 150.5mg of tablet powder, shake the powder in ca and then adjust the volume to 250ml with more acetic acid (0.05M)
5. Filter about 50ml of the solution into a conical flask and then transfer 25 ml of filtrate to 100ml volumetric flask and adjust the volume to 100ml
6. Take 10ml of the diluted extract and transfer to a further 100ml volumetric flask and makeup to volume with 0.05M acetic acid
7. Analyse the standard solutions and the extract from step 6 using the following chromatographic conditions: C-18: mobile phase= 0.05 acetic acid/acetonitrile (90:15); detection: UV- 243nm; flow rate 1ml/min

Question 12

Data obtained from the analysis of paracetamol solution

Answer 12

Question 13

Paracetamol example

Question 14

Main steps to consider

Answer 14

1. Chose a chromatographic method (HPLC or GC)
2. Chose a Column
3. What kind of detector
4. Solvent system
5. Wavelength
6. Chose internal standard
7. Evaluate the sensitivity of the assay
8. Pre-treatment of the samples
9. Analysis

Question 15

1. which chromatographic method would you use

Advantages and disadvantages

Answer 15

Question 16

2. what kind of column

Answer 16

• Reverse or normal phase
• C-18 or C8

Question 17

3. what kind of detector

Answer 17

• Which one to use depends on the structure of the drugs

Using UV detection

• The drug must have reasonable chromophore for UV detection

Question 18

4. what solvent system

Answer 18

• Reverse phase= aq base organic solvents
• Normal phase= organic solvents
• If the drug being analysed can be ionised we must buffer the solution

Question 19

5. what wavelength to use

Question 20

6. which IS

Answer 20

• Commercially available
• Similar chemical and physical properties
• Likely to be presenst in the clinical samples? if yes, dont use it

Question 21

7. Evaluate sensitivity of the assay

Answer 21

• HPLC after parameters have been optimised
• Minimum amount of drug detectable: 10ng
• Signal/noise: >3/1

Question 22

8. Pre-treatment of the samples (to clean and concentrate)

Answer 22

• Use of plasma: how are you going to clean up the sample?

1. Chromatography
   
   • Ion exchange may be difficult- not strongly ionic
   • Preliminary RP chromatography e.g. SEP-PAK (solid phase extraction

Question 23

solid phase extraction

Answer 23

• An efficient method for isolating and concentrating solutes from large volumes of liquid
• Very effective, even when the solutes are present in very dilute concentrations (ppb)
• Materials obtained can be used for chromatographic separation, spectroscopic examination or biological assessment

Question 24

The typical procedure for solid phase extraction

Answer 24

1. Conditioning solvent- (sorbent)
2. Analyte + sample matrix
3. Sorbent + analyte + impurities
4. Washing solvent- sorbent and analyte in extractor and impurities taken out
5. Eluting solvent- analyte removed

Question 25

A typical procedure for solid phase extraction

Answer 25

1. Conditioning the column: wash the column with a 5-10 bed volume of the solvent (same as to be used to elute the analyte
2. Loading the analyte (liquid sample)
3. The sample solvent passes through the column, the analyte + impurities absorbed on the solid phase
4. Wash the column with a solvent, which will elute impurities but leave the analyte on the column
5. Elute the analyte with an appropriate solvent to elute out the analyte (drug)

Question 26

B. extraction

Answer 26

• Extraction with organic solvents
• Try light ones first (organic phase stays on top of aqeous layer e.g. hexane, diethyl ether, ethyl acetate
• Effect of pH on extraction

1. Take 1 ml of buffer with drug and IS
2. Add 3ml of diethyl ether
3. Vortex for 30 sec

Question 27

If the recovery is satisfactory, then go through the same procedure using control plasma instead of buffer

Answer 27

Question 28

How can we get over the problem- back extraction

Answer 28

Question 29

Interfering compounds

Answer 29

• Are there other drugs which the patient may be taking which could interfere with your assay

1. Talk to the clinician about this
2. If there are:
   
   • Run through these drugs with control plasma or
   • Get plasma from patients who are not taking these drugs

• Also, check for interference with the internal standard

Question 30

Development of chromatographic assay (main steps)

Answer 30

1. Chose a chromatographic method
2. Chose a column
3. What kind of detector
4. Solvent system
5. Wavelength
6. Chose IS
7. Evaluate sensitivity of the assay
8. Pre-treatment of the samples
9. Analysis

Question 31

Validation of analytical procedures- methodology

Answer 31

• The extent of validation will depend on where a drug is in the development process
• In USA(FDA)
  
  • Investigational new drug application- application for carrying out clinical trials
  • New drug application- application for sale and marketing of a new drug
• In UK (MHRA)
  
  • Clinical trial authorisation application (for clinical trials)
  • Marketing authorisation application (for sale and marketing)