Chapter 1: Introduction
1.5 Drugs of Abuse Analysis Techniques
1.5.2 Confirmatory Analysis
The immunoassay is a test that is considered to be the first line of defence in the fight against illegal substances. However, because it is only a presumptive test, it needs to be followed up with a test that can confirm the results. In order to establish beyond a reasonable doubt the existence of a prohibited substance, the samples in question ought to be analysed in a testing facility (Lillsunde, 2008). In most cases, conducting this analysis requires a multi-stage procedure, the first stage of which involves the individual compounds being separated using appropriate methods like gas chromatography (GC) or liquid chromatography (LC). The latter is utilized most frequently in the field of forensic toxicology and therefore was the focus of this study. After the separation has been
completed, a combined detector, such as a mass spectrometer (MS), is utilized to detect
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the compounds of interest by comparing them to a set of reference materials (Alhefeiti et al., 2021).
1.5.2.1 Direct Analysis in Real Time-Mass Spectrometry (DART-MS)
In 2005, the DART ion source was made available to the scientific community (Y. Shen, Wu, & Guo, 2016). This source employs a mechanism for ionization that takes place in the gas phase, and it is applicable for the examination of solids, liquids, and gases. Because the sample is directly analyzed on a surface such as paper or glass, there is no need for an extraction process to be performed on the sample, which ultimately results in a reduction in the amount of time required for the analysis. DART can be combined with either the MS detector or a high-resolution time-of-flight mass
spectrometer to obtain a rapid qualitative or quantitative analysis of a wide variety of substances (Lesiak & Shepard, 2014).
In comparison to the techniques that are currently in use, DART-MS has the potential to provide an informative, sensitive, and speedy analytical screening technique.
This could make it possible to conduct rapid screenings of unidentified drugs that are frequently received in forensic laboratories (Drury, Ramotowski, & Moini, 2018).
DART has been put to use 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).
1.5.2.2 Liquid Chromatography-Time-of-Flight Mass Spectrometry (LC-TOF-MS) TOF is a high-resolution method that has become increasingly popular in recent years for high-throughput toxicological screening of unidentified samples. It is common practice for an LC/MS system to be accompanied by either a time-of-flight (TOF) or quadrupole time-of-flight (Q-TOF) analyzer. It has detection limits that can go as low as one part per million (ppm), which contributes to its high level of accuracy in mass measurements. It offers exceptional full-scan sensitivity and comprehensive drug screening, both of which can be accomplished without the use of predefined target analytes (Tsai et al., 2013). The use of exact monoisotopic masses and isotopic configurations in the identification process is made possible thanks to the benefit that high mass accuracy provides. This enables the detection of uncommon metabolites that
43 have standards that are difficult to acquire. In addition, screening for any drugs of
interest in samples that have already been analyzed does not require re-analysis because the results of the TOF-MS test can be reprocessed (Marin, Hughes, Lawlor, Clark, &
McMillin, 2012).
When only using accurate mass and isotopic pattern matching for identification, this method of drug abuse screening has been found to produce unavoidable false- positive data. This is one of the drawbacks that has been observed in relation to this technique. However, relying solely on the examination of fragmentation spectra is an effective method for cutting down on false-positive results. Pelander, Kolmonen,
Ojanpera, and Gergov et al. (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).
1.5.2.3 Ambient Ion Sources for Direct Forensic Analysis
Field-capable mass spectrometers based on membrane inlet systems and hybrid GC systems have been developed and made available in the market in order to cater to the requirements of the forensic and security communities. Recent advancements in ambient ionization mass spectrometry have made it possible to eliminate the need for the time, equipment, and expertise normally associated with sample preparation. As a result, these methods are particularly attractive for on-site analysis. The on-site analysis of seized drugs, explosives, chemical warfare agents, and other compounds of interest could be made possible through the development of fieldable mass spectrometry systems, with an emphasis on commercially available systems (Evans-Nguyen et al., 2021).
1.5.2.4 Gas Chromatography-Mass Spectrometry (GC-MS)
Before being put through the GC-MS instrument, the drugs are first extracted from the sample using the appropriate technique. The sample may also require a derivatization step in order to become volatile and ready to be analyzed in GC-MS.
Ionizing molecules typically involves employing the electron ionization (EI) technique
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with an energy setting of 70 eV. The unique fingerprints of ions are determined for the purpose of identifying compounds by utilizing the resulting mass to charge ratios in conjunction with their abundance. Several GC-MS methods were developed in order to facilitate the analysis of drugs. Ionization via chemical means is utilized in order to bring about the production of fragments that are both less extensive and more stable. Chemical ionization that generates negative ions, in contrast, produces fewer ions that have high levels of sensitivity but lower selectivity. When conducting a confirmation analysis, GC- MS is typically run in the selected ion monitoring mode, and the deuterated internal standards are frequently employed (Lillsunde, 2008).
The GC-MS instrument has several benefits, the most important of which are its affordability and its durability. Since the majority of drug concentrations in certain biological samples appear to be significantly low, additional testing methods that are significantly more sensitive are required (Breidi, Barker, Petróczi, & Naughton, 2012).
GC-MS has been shown by a number of different studies to be effective in the detection of a wide variety of benzodiazepine (BZD) drugs, including oxazepam, lorazepam, bromazepam and diazepam with LOQ 0.52–58.47 ng/ml. GC-MS offers an advantage of quantification and formal identification of BZDs with often low detection limits. However, the use of GC poses a number of problems mainly related to the thermolability of most BZDs that degrade rapidly in the absence of prior derivatization (Qriouet et al., 2019).
GC-MS has been also used for the qualitative and quantitative determination of opioids in the blood and urine such as (tramadol, dextromethorphan, morphine, codeine, 6-acetylmorphine and nalbuphine) in a selected ion monitoring (SIM) mode; with the linearity range for 6-acetylmorphine as 5–150 ng/ml, while for all other target opioids it was 10–1500 ng/ml (Yasien et al., 2022).
1.5.2.5 Liquid Chromatography-Mass Spectrometry (LC-MS/MS)
Electrospray ionization is the most commonly used method for ionization in LC- MS drug analysis (ESI). The Atmospheric Pressure Chemical Ionization (APCI) is an additional ionization process. Several problems with the enhancement and suppression of ions have been reported, but there can also be issues with APCI. Four sample preparation procedures, namely dilution, direct injection, solid-phase extraction, and protein
45 precipitation, were used to pre-treat samples prior to analysis by LC-ESI-MS/MS and LC-APCI-MS-MS. Despite the fact that the matrix effect was observed in both types of ionization, ESI was more susceptible (Lillsunde, 2008).
Oiestadt et al. conducted a screening of 32 substances using simple and rapid liquid–liquid extraction with LC-MSMS. Even though the benzoylecgonine extraction recovery was as low as 0.2%, LC-tandem mass spectrometry was able to test and identify benzoylecgonine (Alhefeiti et al., 2021; Shah, Petroczi, Uvacsek, Ránky, &
Naughton, 2014).
In addition, LC-MS/MS has been commonly used in the forensic laboratory for the identification and quantitation of opiate drugs. For instance, codeine, morphine, 6- acetylmorphine, hydrocodone, hydromorphone, oxycodone, and oxymorphone have been extracted from urine and oral fluid samples and detected using LC-MSMS with LOD 1.5 ng/ml and 99.3% recovery (Pratiwi et al., 2021). Table 8 represents an overview of LC- MS characteristics used in the detection of various drugs.
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Table 8: Chromatographic system characteristic for drugs of abuse using LC-MS/MS
Analyte Matrix Method
Elution Type and Mobile Phase/Flow Rate
Column/
Temperature Detector LOD/
LOQ
RSD/
Recovery
%
Ref.
Morphine Codeine Thebaine Papaverine Noscapine
Pericarpium papaveris (Papaver somniferumL.) in hot pot broth
UPLC- QqQ-
MS
Gradient elution;
methanol (solvent B)
and water (solvent A);
0.3 mL/min
Acquity BEH C18 column; 40
°C
MS in positive electrospray
ionization with multiple reaction monitoring
(MRM)
LOD = 40 ng/kg LOQ = 100
ng/kg
RSD = 16.9–20.5 Recovery =
78.9–124
(Guo, Zhang, Zhao, &
Shao, 2013)
Codeine Guaiphenesin Chlorpheniramine
Ephedrine
Beagle dog plasma
LC- MS/MS
Formic acid: 10 mM ammonium
acetate/
methanol (0.2:62:38,
v/v); 0.2 mL/min
Phenomene x Luna C18
analytical column;
MS- selected reaction monitoring
(SRM) mode
LLOQ = 0.08 ng/mL
RSD = 7 Recovery =
91
(Hu, Zou, Tian, Sun, &
Zhang, 2011)
Codeine Morphine 6-acetylmorphine
Hydrocodone Hydromorphone
Oxycodone Oxymorphone
Neat oral fluid LC- MS/MS
Mobile phase was
initially 95% 5 mM ammonium formate in water with 0.1% formic
acid, decreased to
85% over 2 min then 5% over 1.5
min; 0.5 mL/min
Agilent Poroshell 120 SB-C18
column;
MS-positive electrospray ionization (ESI) mode
LOD = 0.04 ng/mL LLOQ 1.5
ng/mL
RSD = 3.7 Recovery =
99.3
(Grabena uer et al.,
2018)
Codeine Morphine Hydrocodone Hydromorphone
Oxycodone 6-Acetylmorphine
Urine, serum, plasma, whole blood, and meconium
LC- MS-MS
Isocratic acetonitrile
and 2mM ammonium
formate buffer at pH
3.0.
(15%:85%)
0.525 mL/min
MS in positive electrospray
ionization with multiple reaction monitoring
(MRM)
LOD = 1 LLOQ = 2
ng/mL in urine, serum/plas
ma, and whole blood; ng/g
in meconium
Recovery = 90.8 in urine and
50.4 in meconium
(Coles, Kushnir,
Nelson, McMilli n, &
Urry, 2007)
47 Table 8: Chromatographic system characteristic for drugs of abuse using LC-MS/MS (Continued)
Analyte Matrix Method
Elution Type and Mobile Phase/Flow
Rate
Column/
Temperature Detector LOD/
LOQ
RSD/
Recovery %
Ref.
Morphine Codeine
Poppy seed
LC- MS/MS
Gradient program;
mobile phase A (water, 20 mM ammonium hydrogen carbonate,
adjusted with ammonia to pH 9) and mobile phaseB (water/methanol 5:95 (v/v), 20 mM
ammonium hydrogen carbonate,
adjusted with ammonia to pH 9);
0.2 mL/min
Reversed- phase Phenomenex, RP 18 Gemini column; 40 °C
MS-positive electrospray ionization (ESI) mode
LOD = 300,000 ng/kg
Precision = 7.4-9.0 Accuracy =
9.8–17.6
(Sproll, Perz,
&
Lachenmeier, 2006)
LC: liquid chromatography; LOD: limit of detection; LLOQ: lower limits of
quantification; LOQ: limit of quantification; MS: mass spectrometry; PDA: photo diode array; QqQ-MS: triple quadruple mass spectrometry; RP: reversed-phase; UPLC: ultra- performance liquid chromatography.
Certain drugs, like those that are present in low concentrations, can be difficult to analyze using GC-MS techniques. GC-MS has the disadvantage of not being as sensitive as its counterpart, the LC-MS/MS. As a result, the LC-MS analysis calls for a sample with a lower volume. In addition, this instrument has the capability of identifying
substances with a low recovery rate. In addition, LC enables the detection of compounds that are either thermally unstable or polar. LC-MS techniques are especially
advantageous due to their simple sample pre-treatment, as they do not require complex derivation steps or sample cleaning procedures (Shah et al., 2014). Studies reported that LC-MS/MS is the technique of choice for BZD analysis and quantification, compared with the GC method, as it does not expose molecules to thermal degradation and thus makes it possible to overcome derivatization reactions (Qriouet et al., 2019). Thus, all the above listed features of the LC-MS/MS instrument made it the perfect choice to be used for this study.