stability studies of amphetamine and ephedrine derivatives in urine
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Stability studies of amphetamine and ephedrine derivatives in urine. อาจารย์ที่ปรึกษา อ . ดร . พัลลพ คันธิยงค์ นางสาวดรุณรัตน์ แก้วมูล รหัสนักศึกษา 51312305. Stability studies of amphetamine and ephedrine derivatives in urine By C. Jim enez , R. de la Torre - PowerPoint PPT PresentationTRANSCRIPT
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1
Stability studies of amphetamine and ephe
drine derivatives in urine
อาจารย์�ที่ปร�กษา อ.ดร.พั�ลลพั คั�นธิ�ย์งคั�
นางสาวดร�ณร�ตน� แก�วมู ล รหั�สน�กศึ�กษา 51312305
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Stability studies of amphetamine and ephedrin
e derivatives in urine
By C. Jim enez , R. de la Torre
M. Ventura ,J. Segura ,R. Ventura
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Introduction
Knowledge of the stability of drugs in biological fluids is critical for proper interpretation of
analytical results
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• thermal
• chemical
• degradation, matrix degradation
• metabolism, hydrolysis
• transport,
• handling or sample
• storage conditions
Lossesof analys
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Stability testing can be used
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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
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DHHS Guidelines for Federal Workplace Drug Testing
(USA) 1998
retain all confirmed drug positive urine
least 1 year
in frozen storage
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Introduction
None psychostimulants like
ephedrine derivatives.
Ephedrine / amphetamine derivatives
are included in the list of prohibited substances
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Sample
• ephedrine derivatives
ephedrine, norephedrine, methylephedrine, pseudoephedrine, and norpseudoephedrine
• amphetamine derivatives
(amphetamine, methamphetamine, 3,4-methylenedioxyamphetamine (MDA),
and 3,4-methylenedioxymethamphetamine (MDMA))
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Sample
Sterile sample– Long term– Shot term
Non Sterile sample
(MDMA and methamphetamine)– Long term– Shot term
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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.
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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.
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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
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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
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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
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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.%)
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Calculations
• The Dixon’s test (α = 5%)
detect outliers in the replicates (n = 5) of each aliquot
• ANOVA test (α = 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).
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Analysis of ephedrine derivatives
gas chromatograph (HP 5890 series II)
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Analysis of amphetamine derivatives
HP 6890 series gas chromatograph
HP 5973 mass selective detector
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Results
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• coefficients (r 2) up to 0.990 in all calibrations• ephedrine derivatives LOQ 0.4 u g/mL - 27 ug/mL• 50% 70%.
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• coefficients( r 2 ) up to 0 .9 9 0• amphetaminederivativesLOQ71.0ng/mL- 834. ng/mL• uuu uuuuuuuuuuuuuuu uuu uuuuu>60% ,>90%.
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Precision and accuracy
• Both methods good precision and accuracy
• <20 % for the low-concentration control urine samples
• <15 % for the medium and high concentration control urine samples.
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adsorption of the analytes on the sterilizing filter
3210
not statistically significant (p > 0.05) (data not shown), indicating that all the sample batches prepared for stability testing were homogeneous.
4
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Long-term stability
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-10 %
-12 - 5 %
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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 < 0.05)
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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.
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Hughes et al.
• reported t he stability of amphetamine and methamphetamine in spiked urine samples stored at 4 ◦C for up to 6 months
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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
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Dugan et al
studied the stability in clinical samples tested
before and after 1 year of storage at −20 ◦C, [8]
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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 accord
ance with those obtained by
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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.
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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
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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
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• The study demonstrates the feasibility of• preparing certificate reference materials of successfully s
tudied• analytes. This is of special interest for those analytes for
which a• cut-off concentration has been established as positivity c
riterion• for reporting adverse analytical findings, such as amphet
amine• derivatives in drugs of abuse testing, and ephedrine, met
hylephedrine• and cathine in antidoping control.
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