Stability studies of
amphetamine and ephedrine derivatives
in urine
อาจารย์ ท่ ปี รึกษา อ.ดร.พัลลพ คันธิยงค์
นางสาวดรุ ณรัตน์ แก้ วมูล
รหัสนักศึกษา 51312305
1
Stability studies of
amphetamine and ephedrine
derivatives in urine
By
C. Jim enez , R. de la Torre
M. Ventura ,J. Segura ,R. Ventura
Introduction
Knowledge of the stability of
drugs in biological fluids is
critical for proper interpretation
of analytical results
•
•
•
•
•
•
•
thermal
chemical
degradation, matrix degradation
metabolism, hydrolysis
transport,
handling or sample
storage conditions
Losses
of analys
Stability testing can be used
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
DHHS Guidelines for Federal
Workplace Drug Testing
(USA) 1998
retain all confirmed drug positive urine
least 1 year
in frozen storage
Introduction
None psychostimulants like
ephedrine derivatives.
Ephedrine / amphetamine derivatives
are included in the list of prohibited
substances
Sample
• ephedrine derivatives
ephedrine, norephedrine, methylephedrine,
pseudoephedrine, and norpseudoephedrine
• amphetamine derivatives
(amphetamine, methamphetamine, 3,4methylenedioxyamphetamine (MDA),
and 3,4-methylenedioxymethamphetamine (MDMA))
Sample
Sterile sample
– Long term
– Shot term
Non Sterile sample
(MDMA and methamphetamine)
– Long term
– Shot term
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.
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.
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
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
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
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.%)
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).
Analysis of ephedrine derivatives
gas chromatograph (HP 5890 series II)
Analysis of amphetamine derivatives
HP 6890 series gas chromatograph
HP 5973 mass selective detector
Results
•
•
•
coefficients (r2) up to 0.990 in all calibrations
ephedrine derivatives LOQ 0.4 ug/mL - 2.7 ug/mL
recoveries between 50% and 70%.
• coefficients (r2) up to 0.990
•amphetamine derivatives LOQ 71.0 ng/mL - 83.4 ng/mL
• recoveries >60% for amphetamine and MDA, and >90% for methamphetamine and MDMA.
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.
adsorption of the analytes on the sterilizing filter
0
1
2
3
4
not statistically significant (p > 0.05) (data not shown),
indicating that all the sample batches prepared for
stability testing were homogeneous.
Long-term stability
-12 - 5 %
-10 %
MDA 4 C 4%
Shot -term stability
Long-term stability
did not show a significant decrease (p < 0.05)
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.
Hughes et al.
• reported t he stability of amphetamine and
methamphetamine in spiked urine
samples stored at 4 ◦C for up to 6 months
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
Dugan et al
studied the stability in clinical samples
tested
before and after 1 year of storage at −20 ◦C,
[8]
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
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.
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
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 drugtesting and antidoping
• control laboratories
• 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.
ความเห็นที่มีต่องานวิจยั
• สามารถนาไปใช้ ในการเลือกสภาวะในการเก็บรักษาสิ่ งส่ งตรวจที่
เหมาะสม สาหรับการนามาตรวจวิเคราะห์ ซ้า
ปัจจุบันเครื่อข่ ายนิติพษิ วิทยา แห่ งประเทศไทยมีเกณฑ์ การเก็บรักษาสิ่
ส่ งตรวจเพือ่ ตรวจวิเคราะห์ ซ้า โดยกาหนดให้ มีระยะเวลาในการเก็บรักษาสิ่ ง
ส่ งตรวจไม่ น้อยกว่ า 6 เดือน
ความเห็นที่มีต่องานวิจยั
• นาไปใช้ประกอบการพิจารณาแปลผลการตรวจวิเคราะห์
• เพิม่ ความน่าเชื่อถือของผลการตรวจวิเคราะห์
• เป็ นตัวอย่างที่ดีของห้องปฏิบตั ิการที่แนะนาให้ใช้ reference
materials เพื่อความน่าเชื่อถือของผลการตรวจวิเคราะห์ และ
สามารถอ้างอิงได้