Stability studies of amphetamine and ephedrine derivatives in urine

1. Stability studies of amphetamine and ephedrine derivatives in urine อาจารย์ที่ปรึกษา อ.ดร.พัลลพคันธิยงค์ นางสาวดรุณรัตน์ แก้วมูล รหัสนักศึกษา 51312305

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 Nonepsychostimulants 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

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 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).

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

22. coefficients (r2) up to 0.990 in all calibrations • ephedrine derivatives LOQ 0.4 ug/mL - 2.7ug/mL • recoveries between 50% and 70%.

23. 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.

24. 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.

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

27. Long-term stability

28. -12 - 5 % -10 %

29. MDA 4 C 4%

30. Shot -term stability

32. Long-term stability

33. did not show a significant decrease (p < 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. ความเห็นที่มีต่องานวิจัยความเห็นที่มีต่องานวิจัย • สามารถนำไปใช้ในการเลือกสภาวะในการเก็บรักษาสิ่งส่งตรวจที่เหมาะสม สำหรับการนำมาตรวจวิเคราะห์ซ้ำ ปัจจุบันเครื่อข่ายนิติพิษวิทยา แห่งประเทศไทยมีเกณฑ์การเก็บรักษาสิ่ ส่งตรวจเพื่อตรวจวิเคราะห์ซ้ำ โดยกำหนดให้มีระยะเวลาในการเก็บรักษาสิ่งส่งตรวจไม่น้อยกว่า 6 เดือน

44. ความเห็นที่มีต่องานวิจัยความเห็นที่มีต่องานวิจัย • นำไปใช้ประกอบการพิจารณาแปลผลการตรวจวิเคราะห์ • เพิ่มความน่าเชื่อถือของผลการตรวจวิเคราะห์ • เป็นตัวอย่างที่ดีของห้องปฏิบัติการที่แนะนำให้ใช้ reference materials เพื่อความน่าเชื่อถือของผลการตรวจวิเคราะห์ และสามารถอ้างอิงได้