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Stability studies of amphetamine and ephedrine derivatives in urine

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Title: Stability studies of amphetamine and ephedrine derivatives in urine


1
Stability studies of amphetamine and ephedrine
derivatives in urine
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2
  • Stability studies of
  • amphetamine and ephedrine derivatives in urine
  • By
  • C. Jim enez , R. de la Torre
  • M. Ventura ,J. Segura ,R. Ventura

3
Introduction
  • Knowledge of the stability of drugs in
    biological fluids is critical for proper
    interpretation of analytical results

4
  • thermal
  • chemical
  • degradation, matrix degradation
  • metabolism, hydrolysis
  • transport,
  • handling or sample
  • storage conditions

Losses of analys
5
  • Stability testing can be used

6
Stability testing can be used
  • explain discrepancies between reanalyses long
    after initial analyses
  • determine time limits that must be imposed
    between the collection and analysis of samples
    for pharmacokinetic studies
  • identify the optimal storage conditions

7
DHHS Guidelines for Federal Workplace Drug
Testing(USA) 1998
  • retain all confirmed drug positive urine
  • least 1 year
  • in frozen storage

8
Introduction
  • None psychostimulants like
  • ephedrine derivatives.
  • Ephedrine / amphetamine derivatives
  • are included in the list of prohibited substances

9
Sample
  • ephedrine derivatives
  • ephedrine, norephedrine, methylephedrine,
    pseudoephedrine, and norpseudoephedrine
  • amphetamine derivatives
  • (amphetamine, methamphetamine,
    3,4-methylenedioxyamphetamine (MDA),
  • and 3,4-methylenedioxymethamphetamine (MDMA))

10
Sample
  • Sterile sample
  • Long term
  • Shot term
  • Non Sterile sample
  • (MDMA and methamphetamine)
  • Long term
  • Shot term

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12
Sterile samples
  • Long-term stability
  • 4 ?C and -20 ?C / at - 80 ?C as reference
  • 1, 2, 3, 6, 10, 12, 18 and 24 months.
  • Short-term stability
  • 37 ?C / at -20 ?C for comparison purposes.
  • 3 and 7 days.

13
non Sterile samples
  • Long-term stability
  • 4 ?C and -20 ?C / at initial concentration as
    reference value for comparison
  • 6 months
  • Short-term stability
  • 37 ?C / at -20 ?C for comparison purposes.
  • 7 days.

14
Preparation of Sterile samples
  • Blank urine 2 ml sodium azide (0.1, w/v)
  • clarified by filtration (three different filters)
  • spiked standard
  • distributed in aliquots under sterile conditions

15
Preparation of non Sterile samples
  • Blank urine 2 ml sodium azide (0.1, w/v)
  • clarified by filtration (three different filters)
  • spiked standard
  • distributed in aliquots under sterile conditions
    in a laminar

16
Samples analyzed
  • Calibration samples were prepared in duplicate
  • A control sample (three replicates) was analyzed
    in
  • each analytical batch
  • replicates of each aliquot of sample
  • were analyzed at random in the analytical batch

17
Precision and accuracy
  • three different concentration
  • three replicates of control urine samples
  • Precision was expressed as the relative
    standarddeviation (R.S.D.)
  • accuracy was expressed as the relative standard
    error (R.E.)

18
Calculations
  • The Dixons test (a 5)
  • detect outliers in the replicates (n 5) of
    each aliquot
  • ANOVA test (a 5)
  • Homogeneity, adsorption of the analytes on the
    sterilizing filter and stability compare
    concentrations obtained at each storage condition
    with a reference value (concentration of aliquots
    of sample stored at the reference condition).

19
Analysis of ephedrine derivatives
gas chromatograph (HP 5890 series II)
20
Analysis of amphetamine derivatives
  • HP 6890 series gas chromatograph
  • HP 5973 mass selective detector

21
  • Results

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  • coefficients (r2) up to 0.990
  • amphetamine derivatives LOQ 71.0 ng/mL - 83.4
    ng/mL
  • recoveries gt60 for amphetamine and MDA, and
    gt90 for methamphetamine and MDMA.

24
Precision and accuracy
  • Both methods good precision and accuracy
  • lt20 for the low-concentration control urine
    samples
  • lt15 for the medium and high concentration
    control urine samples.

25
adsorption of the analytes on the sterilizing
filter
3
2
4
1
0
not statistically significant (p gt 0.05) (data
not shown), indicating that all the sample
batches prepared for stability testing were
homogeneous.
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  • Long-term stability

28
-12 - 5
-10
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MDA 4 C 4
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  • Shot -term stability

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  • Long-term stability

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did not show a significant decrease (p lt 0.05)
34
Discussion
  • Moody et al.
  • no significant change in analytes concentration
    for up to 17 months. Other studies have also
    demonstrated the stability of these drugs in
    non-preserved urine at different time and
    temperature conditions.

35
Hughes et al.
  • reported t he stability of amphetamine and
    methamphetamine in spiked urine samples stored at
    4 ?C for up to 6 months

36
Discussion
  • For long-term stability
  • statistically significant only observed for the
    ephedrine derivatives at some of thestorage
    conditions tested
  • not exceed the intra-assay precision of the
    corresponding analytical methods

37
Dugan et al
  • studied the stability in clinical samples tested
  • before and after 1 year of storage at -20 ?C, 8

38
Paul et al.
  • investigated the effect of freezing (at -16 ?C to
    -18 ?C) on the concentration of amphetamine and
    methamphetamine in spiked urine samples stored
    for 45 days. In the same way, our observations
    are also in accordance with those obtained by

39
Clauwaert et al.
  • who demonstrated the stability of MDMA and MDA in
    non-preserved urine samples stored at -20 ?C, 4
    ?C and 20 ?C for 21 weeks.

40
ephedrine and amphetamine derivatives
  • sterile samples
  • can be stored at the least. 4 ?C
  • for up to 24 months for
  • non-sterile samples
  • can be stored at the least. 4 ?C
  • for up to 6 months for

41
Discussion
  • The methodology presented when applied to other
    analytes
  • may help to determine optimal storage conditions
    for urine samples
  • to be used as reference materials and for
    positive urine
  • samples that should be retained in drug-testing
    and antidoping
  • control laboratories

42
  • The study demonstrates the feasibility of
  • preparing certificate reference materials of
    successfully studied
  • analytes. This is of special interest for those
    analytes for which a
  • cut-off concentration has been established as
    positivity criterion
  • for reporting adverse analytical findings, such
    as amphetamine
  • derivatives in drugs of abuse testing, and
    ephedrine, methylephedrine
  • and cathine in antidoping control.

43
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44
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