Discuss about the Forensic Toxicology and Drug Analysis.
SPE sample configuration approach is widely utilized in the discipline of forensic toxicology and drug analysis and effectively supersedes other conventional sampling techniques (including liquid-liquid extraction) (1). SPE method continues to benefit the research analysis due to its reduced cost, limited error sources, reduced procedure steps and operation time and utilization of smaller amount of solvent. SPE intervention effectively utilizes the evidence-based approaches including clean-up, purification, concentration and isolation in a versatile manner for retrieving the desirable outcomes (1). The sustained enhancement of stationary phases with appropriate columns is the preliminary cause of the selection of SPE method over LLE (liquid-liquid extraction) intervention (2). SPE intervention is comparatively a faster method that facilitates the systematic tracking of numerous drugs that remained unexplored by the conventional LLE modality (2). Evidence-based research literature reveals the elevated potential of d-SPE (dispersive solid phase extraction) process in terms of evaluating the existence of 13 cytostatic drugs through urine sampling. The high sensitivity of SPE intervention in terms of tracking the drug traces with the lowest quantification limit makes this approach as a preferred diagnostic modality requiring deployment in undertaking forensic drug analysis (3). The analysis by (4) indicates the elevated efficiency of SSE method (in comparison to the routine LLE approach) in evaluating the pattern of urinary morphine. The research intervention by (5)indicates the effectiveness of LC-MS/MS (liquid chromatography tandem mass spectrometry) and solid-phase extraction interventions in terms of attaining sensitive, precise, accurate and rapid resolution of various drugs metabolites in the human urine. Evidence-based analysis by (6) rationally indicates the potential of concomitant utilization of SPE and capillary electrophoresis modalities in terms of evaluating the pattern of drug abuse through urine sample analysis of the drug abusers. The research investigation by (7)reveals the applicability of SPE procedure in term of systematically recovering diuretics at a higher yield. However, the specificity of this intervention is based on the selection of matrix, pre-activated columns and urine sample composition. This research intervention systematically explored the specificity and sensitivity of SPE intervention (as compared to LLE modality) in identifying the pattern of drug traces in human urine samples.
The stock preparation was performed with the systematic utilization of the following ingredients.
- 100ul of 100ug/mL morphine 3-glucuronide (calibrator stock solution)
- 100ul of 100ug/mL morphine 6-glucuronide (calibratorstock solution)
- 100ul of 100ug/ml morphine (calibrator stock solution)
The above-mentioned ingredients were serially incorporated in a screw top test tube (of 10ml capacity and plastic configuration). Subsequently, the volume configuration of 10ml was acquired with the addition of methanol. The lab technician replaced the tip of micropipette while contacting it to different solutions with the objective of reducing the scope of contamination. The tube inversion was eventually undertaken several times with the objective of ascertaining adequate mixing of the desirable solution.
The urine sample of 500ul was prepared in a 10mL test tube (10ml-plastic screw cap type) with the utilization of a micropipette. The following ingredients were subsequently added in the test tube in accordance with the laboratory conventions.
- Internal standard (100uL) [i.e. 1ug.ml morphine-d3 in HCl]
- 1N HCl (1ml)
- Methanol (10uL)
The above-mentioned ingredients were sequentially whirled and vortexed for a duration of one minute. The replacement of the micropipette tips was performed during the systematic preparation of the solutions for reducing the scope of contamination. The freshly labelled plastic screw top (10ml test tube) was utilized for holding the blank urinefollowing its systematic transfer through micropipette (50ul). The following ingredients were subsequently incorporated in the same test tube prior to its 1-min whirling process.
- mlmorphine-d3 in HCl (100ul internal standard)
- 1N HCl (1ml)
- Stock preparation (10uL)
The lab technician replaced the micropipette tip while preparing each solution for avoiding contamination. Both configured test tubes were eventually centrifuged at a speed of 3000rpm, for duration of 5 minutes and at 4°c temperature.
The vacuum manifold rack was utilized for placing both screw top plastic test tubes (10ml each) with the objective of collecting the waste. The waste tubes prepared the ground for setting the taps required for fixating the sample preparation extraction cartridges. The following cartridges were systematically utilized during the process of solid phase extraction.
- MCX3cc cartridge, OASIS
- Waters corporation, Milford, Massachusetts USA
1-ml methanol and 0.1N HCl were subsequently utilized for washing the cartridges. The washing process was undertaken for reducing the scope of dryness of the cartridges. The tap was eventually closed while leaving minimal volume of liquid. The addition of the urine sample to the first cartridge was performed after undertaking the process of centrifugation. The urine sample was instilled in a manner to leave a small amount of the same in the cartridge. The sample of blank urine and stock was then incorporated in the second cartridge while replicating the same process. The micropipette was then utilized for incorporating 0.1N HCl (2ml wash) in cartridges. The sample was again run in a manner for leaving minimal volume in the context of reducing the scope of cartridge dryness. The entire waste was discarded after collecting the same in test tubes (10ml each). A vacuum manifold was utilized for drying the samples at 10mm Hg pressure. The vacuum manifold taps remained accessible for 2 minutes during sample drying. The consistency of pressure was noticed during the entire length of the drying time. Subsequently, both columns were incorporated with 2ml of 5% ammonium hydroxide (menthol base) after the process of drying. The glass test tubes failed to adjust with the vacuum manifold and therefore, plastic test tubes (10ml) were utilized for the eventual running of elute.
Post Laboratory Findings
The laboratory analysis revealed that the weaker solvent passed through the tube under the influence of analyte and the stronger solvent survived in the tube for a longer term (as evident with the elevated retention time peak). The first two urine samples initially displayed elevation in the analyte peak height and area. However, the third sample displayed reduced peak height during the initial phase followed by an abrupt elevation in the analyte peak height and area. Accordingly, the analyte retention time in the initial two samples wascomparatively less than the analyte retention time in the third urine sample.
The analyte peak area and peak height in the first blank urine sample were recorded as the highest in comparison to the other two blank urine samples. Similarly, the analyte retention time in the initial blank urine sample was found to be less in comparison to the analyte retention time in other two urine samples. The IS peak areas and heights in the three samples exhibited the same pattern of variation in comparison to the standardized benchmarks. However, the IS retention time appeared consistent in accordance with the standardized benchmarks in all the three samples. The elevation in analyte peak heights in the samples indicates their elevated purity values (8). The enhancement of analyte peak areas in the samples under the minimum influence of matrix effects indicates the high sensitivity of SPE intervention in tracking the drug traces in the evaluated urine samples (9). The pattern of consistency in the analytes retention times in the three samples indicated the potential of SPE intervention in terms of detecting small concentrations of various drugs particles under standard conditions (10).
The presented laboratory intervention attempted to explore the potential of SPE modality in terms of detecting the quantity of drug traces in three samples of human urine. The recorded variation in the analyte peak area/height/retention time and IS peak area/height/retention time evidentially indicate the elevated affinity of SPE method for the drugs of abuse in the matrix of human urine. This affirms the high precision of SPE approach (in comparison to LLE intervention) in terms of identifying the drugs of intertest in human urine samples with the systematic utilization of mixed mode cartridges. The systematic handling process and precise outcomes make the SPE modality as a preferred laboratory intervention requiring administration for evaluating the pattern of drug abuse during forensic investigation.
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