Using Thin Layer Chromatography (TLC) to Screen Tuberculosis Drugs by obr18219

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									 Using Thin Layer Chromatography
(TLC) to Screen Tuberculosis Drugs

             Kayla Laserson, ScD
         Division of TB Elimination/IA
   Centers for Disease Control & Prevention
• 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
        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
       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
              Field Validation of TLC Kit
                    FDC TB Drugs
• 13 samples from WHO global project on bioavailability of FDC TB
• 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 1999;3:S347-S350
       Performing the TLC Analysis:
             Materials 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
      Performing the TLC Analysis:
•   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
     Performing the TLC Analysis:
        Preparation 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

• Make stock and dilutions
  – Stock solutions and dilutions must be calculated
  – Concentrations normally about 1 mg/mL
     Performing the TLC Analysis:
       Preparation 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
     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
     Performing the TLC Analysis:
            Plate 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)
     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
     Performing the TLC Analysis:
           Spotting 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
       Performing the TLC Analysis:
        Development 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
    Performing the TLC Analysis:
   Visualization 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
  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
      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
Example of TLC Use in the Field
                   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)
• Sample collection from the national TB program or hospital
  pharmacies (Russia, Latvia); local pharmacies (Colombia, India,
  Vietnam); or both (Estonia)
• 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

• 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)
                     Sensitivity and Specificity of TLC

                   UV Confirmatory Method for Rifampin
TLC Method (FDA)

                                     Poor quality Good quality
                                                                      Sensitivity: 4/4 = 100%

                      Poor quality
                                        4              2          6   Specificity: 24/26 = 92%

                      Good quality      0            24          24   Pred. value +: 4/6 = 67%
                                        4            26          30   Pred. value -: 24/24 = 100%
                           Reproducibility of TLC

                            TLC Method (FDA)
TLC Method (CDC)

                                  Poor quality Good quality
                                                                   % agreement:

                   Poor quality      5            14          19   20/36 = 56%

                   Good quality      2            15          17

                                    7             29          36
                    Cost of TLC (1)
• TLC: <1$/test
• 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
                      Cost of TLC (2)
• Cost 2:
   – 67 samples X $1 = $67 for TLC at CDC
   – plus 19 X $30 (confirmatory testing on substandard samples) =
   – = $637

• Potential cost:
   – 67 samples X $30 (confirmatory testing on all samples)
   – = $2010
•   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
•   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

•   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)
     –Allen Kenyon
     –Division of TB Elimination staff
     –TB/Mycobacteriology Branch
• Colombia
• Estonia
• India

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