Emily C. Lennert
morphine, codeine, 6-acetylmorphine, stability, blood, drug, opiate, gas chromatography – mass spectrometry, GC-MS
Papoutsis, I.; Nikolaou, P.; Pistos, C.; Dona, A.; Stefanidou, M.; Spiliopoulou, C.; Athanaselis, S. Stability of morphine, codeine, and 6-acetylmorphine in blood at different sampling and storage conditions. Journal of Forensic Sciences. 2014, 59(2), 550-554.
The opinions expressed in this review are an interpretation of the research presented in the article. These opinions are those of the summation author and do not necessarily represent the position of the University of Central Florida or of the authors of the original article.
Drug stability is important in forensic toxicology, due to possible delays between the time of sample collection and sample analysis. Stability studies are necessary to determine optimal sampling and storage conditions, as well as to determine the length of time for which a sample remains stable. Additionally, forensic laboratories are often required to retain samples after analysis in case of the need for re-analysis at a later date. However, if a drug is unstable, the amount of drug detected in the re-analysis may be different than the amount of drug detected in the initial analysis. Blood samples obtained from heroin abusers generally contain morphine, codeine, and 6-acetylmorphine (6-AM). This study aimed to evaluate the effect of sampling tube type, preservative addition, and anticoagulant addition on the stability of morphine, codeine, and 6-AM in blood samples. Samples were analyzed using a previously validated gas chromatography – mass spectrometry (GC-MS) method for the analysis of morphine, codeine, and 6-AM in blood.
Standards of morphine, codeine, and 6-AM were purchased. Deuterated standards were also purchased to serve as internal standards (IS): morphine-d3, codeine-d3, and 6-AM-d6. All standards were purchased at a concentration of 1 mg/mL. Blood was obtained from drug-free volunteers and was verified as drug free by GC-MS analysis. Working standard solutions were prepared, one containing a mixture of morphine and codeine and their respective IS, and the other containing only 6-AM and its IS. Working solutions were prepared at concentrations of 0.10, 0.20, 0.30, 0.50, 1.50, 3.00, 4.00, 8.00, and 10.0 μg/mL for the opiates, while the IS concentration was held constant at 1.00 μg/mL. Working solutions for the stability study were prepared at 1.0 μg/mL, with one containing morphine and codeine, and the other containing only 6-AM.
Spiked blood samples were prepared by spiking 50 μL of working standard solution into 950 μL of blood. Six calibrator blood samples were prepared for each working solution at concentrations of 5.00, 10.0, 25.0, 75.0, 150, and 500 μg/L. Quality control (QC) samples were also prepared at concentrations of 15.0, 200, and 400 μg/L. For stability studies, samples were prepared at 50 μg/L. The IS concentration in all samples was 50.0 μg/L.
Blood samples were prepared for GC-MS analysis. After spiking, the samples were mixed using a vortex machine. Phosphate buffer was added to the samples, then the samples were centrifuged. Samples were then processed through solid phase extraction columns. The extracted sample was collected and evaporated to dryness, then derivatized by silylation for GC-MS analysis. A more detailed description of the sample preparation is available under Sample Preparation within the study.
The method was validated based on selectivity, specificity, limit of detection (LOD), limit of quantification (LOQ), linearity, precision, accuracy, recovery, and robustness. Stability was studied using the 50 μg/mL samples previously described. Samples were either stored at 4 ˚C, -20 ˚C, or subjected to three cycles of freezing and thawing, i.e. -20 ˚C for 24 hours followed by room temperature for 4 hours. Additionally, three types of storage tube were used: glass, polypropylene plastic (PP), and polystyrene plastic (PS). Additional parameters tested included the addition of preservative (sodium fluoride) and anticoagulant: sodium oxalate or dipotassium ethylene diamine tetraacetic acid (K2EDTA). Additionally, storage time was studied: 4 ˚C for 1 day, 1 week, 2 weeks, and 1 month, and -20 ˚C for 1 week, 2 weeks, 1 month, 2 months, and 3 months. Opiate concentration was determined based on the calibration curves prepared from the calibrator samples.
Selectivity of the method was determined to be acceptable, with limited matrix effects observed in the blank blood samples and no interferences reported. Specificity was also studied and it was reported that, of several other drugs studied, no interferences were observed, allowing for the identification of morphine, codeine, and 6-AM. LOD and LOQ were determined based on the lowest concentration that would yield a signal to noise ratio of 3:1 for LOD and 10:1 for LOQ. LOD and LOQ were reported to be 1.50 μg/L and 5.00 μg/L, respectively, for all opiates. Linearity was acceptable, R2 > 0.996, within the linear dynamic range of the opiates, 5.00 – 500 μg/L. Precision and accuracy were calculated using the QC samples. Precision was reported as percent relative standard deviation, and accuracy was reported as the percent difference between the measured and known concentrations. Results for precision and accuracy were reported in Table 1 within the study. Recovery was also calculated from the QC samples and was reported to be > 94.4% for morphine, 93.4% for codeine, and 94.5% for 6-AM. Robustness was determined by changing parameters such as the pH pf the buffer, derivatization temperature, etc. and the method was determined to be sufficiently robust, as none of the changes produced a statistically significant effect in the results.
Figures 1-6 within the study summarize the effects of the stability studies. Concentration changes after three freeze-thaw cycles were reported to be -3.97%, -10.3%, and -20.8% for morphine, codeine, and 6-AM, respectively. 6-AM exhibited the greatest instability, although each opiate had decreased concentrations in all conditions. Under both storage temperatures, between the same types of tubes, statistically significant differences in the concentration of opiate were observed depending on the presence or absence of preservative. Minor concentration changes for morphine and codeine were observed in PS tubes containing preservative and either anticoagulant at 4 ˚C. In 6-AM samples, minor concentration changes were reported for glass and PP tubes with sodium oxalate as the anticoagulant and preservative. At -20 ˚C, minor concentration loss for all analyte was reported for glass tubes with preservative and sodium oxalate as the anticoagulant. Sodium fluoride preservative significantly improved the stability of samples. 6-AM significantly decreased in concentration when preservative was not added: decreased up to 94.3% at 4 ˚C for 1 month and up to 72.0% at -20 ˚C for 3 months. Anticoagulant type did not show a significant effect on the stability. Loss of analyte was lower in samples stored in glass tubes compared to PP and PS plastic tubes. However, the effect of tube choice became insignificant for morphine and codeine with the addition of preservative.
- 6-AM was shown to be the most unstable of the opiates analyzed.
- Sodium fluoride (preservative) significantly improves the stability of all opiates studied in all conditions.
- The smallest changes in opiate concentrations were observed in samples stored in glass tubes at -20 ˚C.
Optimal sampling and storage conditions must be understood for the most effective forensic toxicological analysis.
Blood samples containing opiates may be best stored in glass tubes at -20 ˚C.