Title: Stability studies of amphetamine and ephedrine derivatives in urine
1Stability 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
3Introduction
- 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
6Stability 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
7DHHS Guidelines for Federal Workplace Drug
Testing(USA) 1998
- retain all confirmed drug positive urine
- least 1 year
- in frozen storage
8Introduction
- None psychostimulants like
- ephedrine derivatives.
- Ephedrine / amphetamine derivatives
- are included in the list of prohibited substances
9Sample
- ephedrine derivatives
- ephedrine, norephedrine, methylephedrine,
pseudoephedrine, and norpseudoephedrine - amphetamine derivatives
- (amphetamine, methamphetamine,
3,4-methylenedioxyamphetamine (MDA), - and 3,4-methylenedioxymethamphetamine (MDMA))
10Sample
- Sterile sample
- Long term
- Shot term
- Non Sterile sample
- (MDMA and methamphetamine)
- Long term
- Shot term
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12Sterile 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.
13non 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.
14Preparation 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
15Preparation 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
16Samples 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
17Precision 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.)
18Calculations
- 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)
20Analysis of amphetamine derivatives
- HP 6890 series gas chromatograph
- HP 5973 mass selective detector
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23- 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.
24Precision 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.
25adsorption 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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27 28-12 - 5
-10
29MDA 4 C 4
30 31(No Transcript)
32 33did not show a significant decrease (p lt 0.05)
34Discussion
- 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.
35Hughes et al.
- reported t he stability of amphetamine and
methamphetamine in spiked urine samples stored at
4 ?C for up to 6 months
36Discussion
- 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
37Dugan et al
- studied the stability in clinical samples tested
- before and after 1 year of storage at -20 ?C, 8
38Paul 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
39Clauwaert 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.
40ephedrine 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
41Discussion
- 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.
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