Using Thin Layer Chromatography TLC to Screen Tuberculosis Drugs

1
Using Thin Layer Chromatography (TLC) to Screen
Tuberculosis Drugs

• Kayla Laserson, ScD
• Division of TB Elimination/IA
• Centers for Disease Control Prevention

2
Definition

• TLC is a 2-dimensional form of chromatography
  that has two components
• A mobile phase (developing solvent) and
• A stationary phase (a plate or strip coated with
  a form of silica gel)
• TLC separates compounds based on polarity
• The TLC kit, as developed by the FDA, provides
  qualitative and semi-quantitative screening of
  drug content

3
Qualities of FDA-Developed TLC Kit Methodology

• Is low cost
• Has short analysis time
• Has easy sample preparation
• Produces spots that can be visualized
• Seldom requires sample cleanup
• Is adaptable to most pharmaceuticals
• Uses small quantities of solvents
• Requires minimal training
• Is reliable and quick
• Uses minimal amount of equipment
• Can use densitometers to increase accuracy of
  spot concentration

4
Field Validation of TLC Kit

• To detect diethylene glycol contamination of
  gylcerine in Haiti
• To determine the quality of paracetamol tablets
  marketed in Bangladesh
• To establish the first drug quality screening
  laboratory in Swaziland
• To screen the quality of fixed-dose combination
  (FDC) TB drugs

5
Field Validation of TLC Kit FDC TB Drugs

• 13 samples from WHO global project on
  bioavailability of FDC TB drugs
• Screened by TLC kit, then analyzed at FDA
• All contained stated drugs
• 5 (38) were substandard - 2 low rifampicin, 1
  high rifampicin, 1 high PZA, 1 rifampicin
  extra spot
• TLC detected both with low rifampicin potency
• TLC detected all 3 with low rifampicin
  bioavailability
• Kenyon T, Kenyon A, Kgarebe B, et al. Detection
  of substandard fixed-dose combination
  tuberculosis drugs using thin-layer
  chromatography.
  Int J Tuberc Lung Dis
  19993S347-S350

6
Performing the TLC AnalysisMaterials Needed

• Solvent bottles 1 liter
• Small bottles, wide mouth 100 mL
• Graduated syringes 1, 5, and 10 mL
• Pestle
• Graduated cylinders 25, 50, and 100 mL
• Small sample vials 1.5 and 6 mL
• Micropipettes 1,2,3,4, and 5 microliters
• Pasteur pipettes and rubber bulb assorted sizes
• Beakers assorted small sizes
• Test tubes 3 to 10 mL sizes
• TLC kits

7
Performing the TLC AnalysisProcedures

• Preparation of sample
• Preparation of standards
• Preparation of developer solvent
• Plate marking
• Spotting of the plate
• Development of plate
• Visualization and interpretation
• Calculations of Rf values
• Acceptance and rejection of sample

8
Performing the TLC AnalysisPreparation of Sample

• Take one dosage unit or a composite of dosage
  units and place in small plastic bag, grind to
  powder, transfer to a suitable vessel, and add
  proper solvent to dissolve active ingredient
• Make stock and dilutions
• Stock solutions and dilutions must be calculated
• Concentrations normally about 1 mg/mL

9
Performing the TLC AnalysisPreparation of
Standards

• Place one reference tablet into a vessel or a
  DiSPO test tube, add sufficient solvent to make a
  solution equivalent to 100 of the active dosage
  strength in the tablets (capsules) i.e., 5 mg/5
  mL
• Take 4 parts of the 100 solution and add 1 part
  solvent to make the 80 standard

10
Performing the TLC Analysis Making Your Own
Standards

• If no reference standard tablets are available, a
  primary or secondary standard must be available
  to make your own
• Weigh the appropriate amount on an analytical
  balance
• Perform proper dilutions to make the appropriate
  concentration equivalent to 100 of the active
  dosage strength in the prepared sample
  formulation (e.g., 5 mg/5 mL)
• Prepare the 80 standard

11
Performing the TLC AnalysisPlate Marking

• The plate is a plastic-backed, silica-coated
  strip
• 5 x 10 cm plates or strips should be provided in
  the kit, or cut from 20 x 20 cm plastic-backed,
  silica-coated sheets
• Mark a line about 1 cm below the top
• Mark a small point on either side of your
  spotting point about 2 cm from the bottom
• Do not remove silica from sides, top, or bottom
• Generally a distance of 10 cm is used as the
  development of a plate so as to make the
  calculation of the Rf value easy
• Rf is defined as the movement of the sample spot
  divided by the movement of the developing solvent
  ( x/10 cm)

12
Performing the TLC Analysis Spotting the Plate

• The sample solute must be in a suitable solution
  for spotting
• If not, the sample solute must be manipulated to
  obtain a suitable solution for spotting

13
Performing the TLC AnalysisSpotting the Plate

• Spot 1 5 microliters of the sample in the center
  of the plate at the origin line
• Spot 2 and 3 5 microliters of the 100 standard
  and the 80 standard on either side of the sample
• Note If 3 microliters of sample is spotted,
  spot 3 microliters of the standards

14
Performing the TLC AnalysisDevelopment of the
Plates

• After spotting, attach the TLC plate to the
  aluminum frame with the clamp and lower it into
  the plastic bag with the fishhook
• Allow the TLC plate to stay in the bag without it
  touching the solvent (the developer is usually a
  mixture of solvents on a parts basis) for about 5
  minutes to reach equilibrium
• Pull the plastic bag down to allow the developing
  solvent to contact the lower 1 cm of the TLC
  strip
• Develop the strip to the top marked line (5
  minutes)
• Stop the development
• Remove the TLC strip, lay it flat, and allow the
  solvent vapors to evaporate

15
Performing the TLC AnalysisVisualization and
Interpretation

• Most pharmaceutically active drugs will not be
  visible to the naked eye
• Spots can be visualized by two basic techniques,
  depending upon the drug being tested
• Ultraviolet light at 254 nm (shortwave UV). Long
  wave UV (340 nm) is used less commonly.
• The sample spots will appear as black spots on a
  fluorescent green background
• Use battery-operated UV lamps
• Staining to make spots visible

16
Performing the TLC Analysis Calculate the Rf
Values

• The Rf value is calculated by measuring the
  distance the sample spot travels divided by the
  distance the developing solvent travels
• Values below 0.1 are considered poor the spots
  are too close to origin
• Values of 0.1 to 0.8 are good and any other spots
  (impurities) or other active compounds are
  resolved from each other
• Above 0.8 poor spots may be too broad or
  distorted

17
Performing the TLC Analysis Acceptance or
Rejection Criteria

• Sample spot has an intensity between the
  standards of 80-100 ACCEPT
• If lower, REJECT
• If higher, re-spot a standard at a concentration
  higher than 100 the 100 standard and the
  sample may be needed to estimate concentration
• Normally, bad drug samples will be lower than
  100 level
• Sample spots should be separated from any other
  active drugs (combination drug products like
  isoniazid INH and rifampin RIF) ACCEPT
• Sample spots should be separated from any
  decomposition products, impurities, or excipients
  in the drug formulation. If many alternate spots
  besides the active are found, the drug may be
  decomposed REJECT
• The sample and standard should have identical Rf
  values ACCEPT

18
Example of TLC Use in the Field
19
Study Objectives

• To perform a preliminary assessment of TLC to
  screen the quality of TB drugs purchased or
  obtained from TB programs in selected areas in
  Colombia, Estonia, and India
• To validate the TLC results using confirmatory
  pharmacopeia methods (HPLC, UV)

20
Methods

• Sample collection from the national TB program or
  hospital pharmacies (Russia, Latvia) local
  pharmacies (Colombia, India, Vietnam) or both
  (Estonia)

21
Methods

• Pharmacies chosen at random from private
  government pharmacies in area
• 10-20 samples collected per specific TB drug
• Single
• Fixed dose combination (FDC)
• Collection included different lots/manufacturers
  sold at pharmacy
• Lot number, manufacturer, and expiration date
  recorded for each TB drug

22
Methods

• CDC performed TLC on INH and RIF
• FDA retested all specimens found to be
  substandard at CDC plus a 10 sample of normal
  specimens
• Qualitatively and semi-quantitatively using TLC
• Quantitatively using HPLC (INH) and UV (RIF)

23
Sensitivity and Specificity of TLC
UV Confirmatory Method for Rifampin
Sensitivity 4/4 100 Specificity 24/26
92 Pred. value 4/6 67 Pred. value -
24/24 100
Poor quality
Good quality
4
6
2
Poor quality
TLC Method (FDA)
24
0
24
Good quality
30
4
26
24
Reproducibility of TLC
TLC Method (FDA)
agreement 20/36 56
Poor quality
Good quality
5
14
19
Poor quality
TLC Method (CDC)
2
15
17
Good quality
36
7
29
25
Cost of TLC (1)

• TLC
• Confirmatory methods 30/test (at FDA lab)
• Cost 1
• 67 samples X 1 67 for TLC at CDC
• plus 19 X 1 (second TLC on substandard samples)
  19
• plus 6 X 30 (confirmatory testing on above
  samples) 180
• TOTAL 266

26
Cost of TLC (2)

• Cost 2
• 67 samples X 1 67 for TLC at CDC
• plus 19 X 30 (confirmatory testing on
  substandard samples) 570
• 637
• Potential cost
• 67 samples X 30 (confirmatory testing on all
  samples)
• 2010

27
Conclusions

• Both sensitivity and specificity of TLC exceed
  90
• 5/7 RIF samples of poor quality were in FDC
  samples
• 1/10 (10) INH samples and 4/30 (13) RIF samples
  abnormal
• TLC can detect substandard TB drugs
• TLC is a convenient, easy, and inexpensive method
  to screen the quality of TB medications in
  settings where routine use of confirmatory
  methods is not practical

28
Recommendations

• Use of TLC to screen for poor TB drug quality,
  with adequate training to ensure
  reproducibility--particularly in areas where
  quality assurance laboratories are less
  accessible or available
• TB drugs that demonstrate poor quality by TLC
  could then be further tested with official
  confirmatory methods such as UV and HPLC
• TLC does not replace the confirmatory methods it
  may be used to help identify which drugs are
  highest priority for testing by the confirmatory
  methods (and to thus save resources)

29
Acknowledgments

• FDA
• Allen Kenyon
• CDC
• Division of TB Elimination staff
• TB/Mycobacteriology Branch
• Colombia
• Estonia
• India