The rapid screening of TB drugs by Thin Layer Chromatography TLC

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The rapid screening of TB drugs by Thin Layer
Chromatography (TLC)

• Alexander Nyarko Ghana
• Annette Bukirwa Uganda
• Patrick Kibiego Kenya
• Daniel Kekwaletswe Botswana
• Margareth Magagula Swaziland
• Mike Green - USA

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INTRODUCTION (1)

• TB is a global problem with a high prevalence in
  Sub-Saharan Africa
  
• TB incidence and prevalence as well as drug
  resistance is increasing
  
• Factors contributing to TB problem
• Poorly managed TB DRUGS
• Poor patient compliance
• Poor quality of TB DRUGS

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INTRODUCTION (2)

• Difficulty of drug management as a result of drug
  donations in some countries
  
• WHO/CDC/FDA and country national laboratories
  have a responsibility of assuring quality of TB
  drugs by developing techniques such as TLC
  
• TLC can help in assessing TB drug quality

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OBJECTIVES

• To learn the TLC technique
• To use TLC to assess the quality of TB drugs
• To confirm the TLC results using alternative
  (USP/BP) methods
  
• Share experiences between participants

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BACKGROUND

• Cause Mycobacterium tuberculosis
• How is it transmitted? airborne
• Natural prognosis of the disease
• 50 die
• 25 spontaneous cure
• 25 chronic excretors

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(No Transcript)
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World TB Situation Estimated incidence of TB 1999

• Country Cases (000S) Rate /100K
• 1 India 1,847 185
• 2 China 1,300 103
• 3 Indonesia 590 282
• 4 Nigeria 327 301
• 9 South Africa 197 495
• 13 Kenya 123 417
• 19 Uganda 72 343
• 21 Zimbabwe 65 562
• Angola 16
  129
  
• Botswana 8 513
• Ghana 10 53
• Lesotho 4 sm
• Swaziland
  2 sm
  

Source Global Tuberculosis Control WHO Report
2001
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DRUG RESISTANT TB

• Resistance to TB drugs is increasing
• Resistance to multidose (MDR) is increasing
• Isoniazid, rifampicin, pyrazinamide, ethambutol,
  streptomycin

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SAMPLING

• Sampling data were recorded
• Manufacturer
• Lot number
• Expiration
• Label claim
• Country where drug was sampled
• Entered into database by CENQAM
• Samples were coded therefore blind to the
  analysts

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PROCEDURE

• Participants performed TLC analysis on test
  drugs
  
• INH
• RIF
• PZA
• ETH
• STR
• Combinations
• Cotrimoxazole also tested used for secondary
  infections associated with HIV
  
• Confirmatory testing performed by CENQAM USING
  UV, HPLC, Dissolution

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MATERIALS

• TLC Kit
• Commonly used solvents
• Reference standards
• Detection by handheld UV lamp battery operated
• Iodine staining
• Visual

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PERFORMING ANALYSIS

• Prepared sample and standards
• Prepared developing solvent
• Mark plate
• Spot plate
• Develop plate
• Dried
• Detected spots
• Compared to std determinedif is within range
• Calculate Rf values
• Accept/Reject

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RESULTS

• Samples from 6 countries
• South Africa Participant countries

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DESCRIPTIVE COUNTRY RESULTS - TLC
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DESCRIPTIVE RESULTS TESTED BY TLC
One failed due to labeling problem
Failure due to apparent solubility problem
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TLC CONFIRMATORY TESTING
Dissolution results
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DISSOLUTION RESULTS
Unofficial test/multiple tablets per vessel
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SUMMARY OF RESULTS

• Out of 29 drugs tested by both UV TLC only 2
  (apparently) did not correlate
  
• We can not call the 2 non correlations failures
  further testing would be needed
  
• 29/29 100 correlation

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LIMITATIONS OF TECHNIQUE

• Must have reference standards not always
  available
  
• The analyte detection is somewhat subjective
• Solvent evaporation may alter results
• For field operation a battery operated detector
  is needed

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STRENGTHS OF TECHNIQUE

• Results reliable
• Specific sensitive
• Easily adaptable
• Rapid
• Mobile
• Easily obtained solvents
• Can spot more than one sample per plate

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DISCUSSION

• To investigate the ability of the analyst to
  detect a bad or defective drug product
  
• Specially prepared sample was made by emptying
  part of a RIF 150mg capsule and replacing the
  material with PZA and sugar remixing, repacking
  the capsule
• The assigned analyst tested for RIF and found
  RIF
  an unknown component.

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CONCLUSION RESULTS 1

• African TB drugs tested
• By TLC the tested samples were found to comply
  with the label claim within the range of 85-115,
  with 3 exceptions
  
• By UV, 4 samples were found to lie outside the
  range unofficial tests
  
• By dissolution, 7 samples apparently failed, but
  these were unofficial dissolution procedures. All
  TB drugs tested using the official dissolution
  procedure passed
• Based on our testing, we found no validated
  failures, therefore the quality of the TB drugs
  appears adequate.
  

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CONCLUSION-RESULTS 2

• No sample had a failure by official testing
• Potential failures would require further testing
• One R/I combination product failure found by TLC
  was due to a confusing label, a potential cGMP
  violation
  

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CONCLUSION - METHOD

• TLC Method
• Works well for initial screening for TB drugs
• Sensitive specific
• Very versatile can be used in lab or field (no
  electricity needed)
  
• Fast, economical
• Reliable/reproducible
• Available for other drugs
• Method modification development is easy

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RECOMMENDATIONS

• WHO should make it an official method with an
  accompanying manual
  
• WHO should facilitate a network amongst
  participants
  
• More seminars training in drug analysis
• Assistance in obtaining reference standards
• Use of technique should be followed up by survey
  of countries which had the training
  
• After TLC screening, it will be important to
  perform dissolution testing
  
• Follow up training on dissolution testing
• Simplified dissolution technique
• Need validated confirmatory test methods for FDC

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ACKNOWLEDGEMENTS

• WHO
• CDC
• CENQAM
• Facilitators
• Participants
• Governments
• Gateway Analytical