stability studies of amphetamine and ephedrine derivatives in urine

1

Stability studies of amphetamine and ephe

drine derivatives in urine

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นางสาวดร�ณร�ตน� แก�วมู ล รหั�สน�กศึ�กษา 51312305

Stability studies of amphetamine and ephedrin

e 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

Lossesof 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, 3,4-methylenedioxyamphetamine (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 (r 2) up to 0.990 in all calibrations• ephedrine derivatives LOQ 0.4 u g/mL - 27 ug/mL• 50% 70%.

• coefficients( r 2 ) up to 0 .9 9 0• amphetaminederivativesLOQ71.0ng/mL- 834. ng/mL• uuu uuuuuuuuuuuuuuu uuu uuuuu>60% ,>90%.

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

3210

not statistically significant (p > 0.05) (data not shown), indicating that all the sample batches prepared for stability testing were homogeneous.

4

Long-term stability

-10 %

-12 - 5 %

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 accord

ance 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 drug-testing and antidoping

• control laboratories

• 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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