Cover: image related to the types of matrices used in the study (photo: by Manal Ali Alhefeiti). Substance abuse is on the rise among the population of the United Arab Emirates (UAE).
Introduction
Overview
If these samples are found above the threshold, they are sent for confirmation in blood, urine and saliva using more sophisticated and time consuming techniques such as Gas Chromatography-Mass Spectrometry (GC-MS) and LC-MS. Although gas chromatography-mass spectrometry (GC-MS) is the method of choice for general unknown analysis, this technique often requires specific chemical derivatization.
Medical Consequences of BZDs, OPI and CAD
- Benzodiazepines
- Opiates
- Combined Acid Drugs
The compounds of BZDs bind to GABA-A receptors and modify the three-dimensional disposition of the receptor (allosteric modulation), which favors the opening of the chloride channel by the action of GABA (Figure 2). This is a result of the potential impairment that can be caused to a person's physical as well as mental abilities (Mets et al., 2011).
Testing Strategies for Drugs of Abuse
- Physical/Behavioral Testing Regimes
- On-site/Roadside Drugs of Abuse Testing
Usually, on-site testing involves the collection of oral fluid or exhaled breath (Alhefeiti et al., 2021). 13 Table 1: Site systems are used for the determination of various types of drugs (continued).
Popular Matrices for DOA Analysis
- Urine Matrix for DOA Analysis
- Blood Matrix for DOA analysis
- Hair Matrix for DOA Analysis
- Saliva DOA Analysis
- Sweat DOA Analysis
Moreover, some of the detected CAD in urine such as PGB and GBP were done by LLE (Almalki et al., 2021). A further significant advantage is that drugs can be identified shortly after consumption, before the processes of metabolism and/or filtration (Mali et al., 2011).
Drugs of Abuse Analysis Techniques
- Immunoassay Techniques for DOA Analysis
- Confirmatory Analysis
In addition, there is insufficient information on the possibility of reabsorption of pollutants from the environment into the skin (Saito et al., 2011). DART has begun to be used in the detection of a wide variety of illicit drugs, including cathinone drug mixtures (Domin, 2013), heroin, LSD, and other new psychoactive substances (Nie et al., 2016). It offers exceptional full-scan sensitivity and comprehensive drug screening, both of which can be achieved without the use of predefined target analytes (Tsai et al., 2013).
Tsai et al., 2013) were the ones who were able to make the application of LC-TOF-MS in the field of drug toxicology a successful endeavor. The LC-TOF-MS technique has been applied to the investigation of various categories of drug standards, including benzodiazepines, opiates, stimulants, antidepressants, synthetic cannabinoids, and cathinone (Guale et al., 2013). However, the use of GC presents a number of problems mainly related to the thermal solubility of most BZDs that degrade rapidly in the absence of prior derivatization (Qriouet et al., 2019).
GC-MS is also used for the qualitative and quantitative determination of opioids in blood and urine such as (tramadol, dextromethorphan, morphine, codeine, 6-acetylmorphine and nalbuphine) in selected ion monitoring mode (SIM); with a linearity range for 6-acetylmorphine of 5–150 ng/ml, while for all other target opioids it was 10–1500 ng/ml (Yasien et al., 2022).
Research Objectives
GC-MS has the disadvantage of not being as sensitive as its LC-MS/MS counterpart. Studies have reported that LC-MS/MS is the technique of choice for BZD analysis and quantification compared to the GC method, as it does not expose the molecules to thermal degradation and thus allows derivatization reactions to be overcome (Qriouet et al., 2019). ). Thus, all the above features make the LC-MS/MS instrument the perfect choice for use in this study.
Identify all compounds and metabolites of BZD, OPI and CAD for which analysis is possible. Optimize and develop new analytical procedures for a select group that are prohibited in the UAE. Develop and validate the analytical procedures for the analysis of drugs in urine/blood and hair samples.
Analysis of multiple drug classes (BZD, OPI, CAD) in urine/blood/hair samples collected from drug users in the UAE.
Methods
Reagents and Standards
Specimen Collection and Pre-treatment Protocols
- Urine and Blood Samples Collection and Pre-treatment
- Hair Samples Collection and Pre-treatment
Hair samples were obtained by cutting strands of hair from the posterior parietal region of the scalp or the back of the skull as close to the scalp as possible with scissors. Samples were collected by a police officer and stored at room temperature in a dry area, then sent to the UAE MOJ-FML, and analysis was performed within 1 year of acquisition.
Specimen Screening Analysis Using Immunoassay Technique
Elevated levels of analytes in a sample assessed by the competitive format will result in reduced binding of the horseradish labeled antigen. Production calibrator standards (9 levels) and quality control levels (low and high) are easily prepared by adding 1 mL of distilled water to the vials. In addition, no sample preparation is required to test suspect samples (urine and blood) by immunoassay.
It only involves taking about 1 ml of your sample and placing it in a sample cup followed by insertion into the immunoassay instrument. The cutoff ranges used in MOJ-FML for urine and blood samples are summarized in Table 10. Samples with negative results in the IA system, i.e. all values below the established cut-off values, will be.
While samples with positive results in the IA system, i.e. any value above the limits, will be followed after the required extraction by a confirmation test using GC-MS or LC-MS/MS (as illustrated in Figure 15).
Specimen Extraction From Urine, Blood and Hair Samples
65 Table 13: Preparation of calibrators for the generation of the calibration curve for the BZDs method from urine and blood matrix. 67 The opiate method for urine and blood matrix involves the preparation of two working calibrator standards (20 μg/mL and 1 μg/mL) (Table 17) for the preparation of selected calibration points (7 levels) as shown in the table 19. For the hair matrix, the preparation of six calibration levels involves the use of 1 µg/mL working standards as shown in Table 20.
The preparation of OPI calibrators can be performed by adding 100 mg blank hair to a disposable glass tube and increasing the different concentrations, followed by the addition of 20 µl IS in each tube. 71 The CAD method for urine and blood matrix included the preparation of 10 µg/ml working calibrator standards (Table 21) for the preparation of selected calibration points as shown in Table 23. The preparation of CAD calibrators can be performed by the addition of 100 µl blank urine or blood in disposable glass tubes and making up the different concentrations according to Table 23, followed by the addition of 20 µl IS in each tube; treat the resulting liquids as samples and then follow the sample preparation step.
The preparation of CAD calibrators can be performed by adding a 100 mg blank hair sample in a disposable glass tube and injecting the different concentrations according to Table 26, followed by the addition of 20 µl of IS in each tube; treat the resulting liquids as samples and then follow the sample preparation step.
Method Development for LC-MS/MS Confirmatory Analyses
Optimization of LC and MS parameters
Optimization of MRM Parameters for Confirmatory Analysis
Method Validation Parameters for Immunoassay Analysis
Method Validation Parameters for LC-MS/MS Analysis
Results and Discussions
Method Validation Results
- Validation Data for Immunoassay
- Validation Data for Confirmatory Test (LC-MS/MS)
The method validation parameters for BZDs, OPI and CAD in the urine matrix were found within an acceptable range for each QC level. For example, the percentage of the coefficient of variation and accuracy indicates that the method used to analyze the parent drugs and their metabolites has good precision and accuracy for intra- and interday. In addition, Table 50-42 illustrates the percentage of intra- and inter-day precision and accuracy results among six quality control samples for each quality control level.
The method validation parameters for BZDs, OPI and CAD in blood matrix were found in an acceptable range for each QC level. For example, the percentage of the coefficient of variations and precision indicates that the method used to analyze the parent drugs and their metabolites has good precision and accuracy for intra- and inter-day. Furthermore, the listed Tables (43, 44 and 45) illustrate the percentage of intra- and inter-day precision and accuracy results among six quality control samples for each quality control level.
For example, the percentage of the coefficient of variation and accuracy indicate that the method used to analyze parent drugs and their metabolites has good intra- and inter-day precision and accuracy.
Urine, Blood and Hair Samples Results for Drugs Users
- Immunoassay Results for Samples Collected from Drug Users
- LC-MS/MS Confirmatory Results
135 Due to the lack of selectivity in the IA instrument, it can cause various false positive and also false negative results. Interestingly, two urine samples obtained from the male suspects were positive for creatinine (lower than 20 mg/dL); however, these were still positive for PGB in one case (above 500 ng/ml) and amphetamine with methamphetamine in another case. Bromazepam, alprazolam, midazolam, nordiazepam, diazepam, oxazepam, 7-aminoclonazepam and clonazepam were detected and confirmed at different ranges in the suspects' urine samples.
In contrast, eight different drugs belonging to the opiate group were found in the urine samples of the suspects. Figures 37 and 38 illustrate the MRM-LC-MS/MS chromatogram of oxazepam and diazepam drugs, respectively, in the case of TOX-U-112. The range of drugs found in the blood matrix and their frequencies are given in Table 52 and Figure 39.
In the results of the tested samples, we noticed that the parent drugs such as tramadol, diazepam and clonazepam were detected in higher concentrations in the blood samples than their metabolites, in contrast to the results obtained from the urine samples. Codeine in case 1 was not detected in the hair matrix, which is explained by its low concentration (M. Shen et al., 2013). In another case, a low concentration of morphine was detected in a urine sample and in a hair matrix.
Conclusion
Future perspectives
All of these may improve our understanding of how these and other bacterial strains can be efficiently used for the degradation of organic pollutants. Simultaneous screening and determination of 128 date-rape drugs in urine by gas chromatography-electron ionization-mass spectrometry. Esi-lc-ms/ms for therapeutic drug monitoring of the binary mixture of pregabalin and tramadol: Human plasma and urine applications.
Validation of the rapid and simple LC-MS/MS method for the quantification of pregabalin in plasma of acutely poisoned patients. Rapid and simple multi-analyte LC–MS/MS method for the determination of benzodiazepines and Z-hypnotic drugs in blood samples: Development, validation and application based on three years of. Clinical and forensic diagnosis of very recent heroin use by 6-acetylmorphine immunoassay testing and LC-MS/MS analysis in urine and blood.
Enzymatic digestion and selective quantification of underivatized delta-9-tetrahydrocannabinol and cocaine in human hair by gas chromatography-mass spectrometry.
