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 Table of Contents  
Year : 2021  |  Volume : 7  |  Issue : 4  |  Page : 123-136

Hyphenated techniques in liquid chromatography and their applications in forensic toxicology: A review

1 Department of Forensic Sciences, College of Criminal Justice, Naif Arab University for Security Sciences, Riyadh, Saudi Arabia
2 Institute of Forensic Medicine, Aachen University of Technology, Aachen, Germany; Royal Clinics King Faisal Specialist Hospital and Research Centre, Royal Toxicological Laboratory, Riyadh, Saudi Arabia

Date of Submission13-Sep-2021
Date of Decision28-Nov-2021
Date of Acceptance01-Dec-2021
Date of Web Publication30-Dec-2021

Correspondence Address:
Sachil Kumar
Department of Forensic Sciences, College of Criminal Justice, Naif Arab University for Security Sciences, Riyadh
Saudi Arabia
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/jfsm.jfsm_65_21

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Conventional analytical methods, such as gas chromatography, high performance liquid chromatography (LC), ultra-violet, and others, are ineffective in addressing the increasing number of problems in forensic toxicology. Hyphenated analytical methods, wherein the separation method are coupled or combined with spectral methods, with the help of a proper interface, are the available alternative options. The key benefits of these methods are the requisites of low limits for detection, shorter analytical time, the possibility of automation, better reproducibility, and high precision and repeatability. This review discusses on some of the hyphenated analytical methods that involve LC as the separation tool, for their most recent applications in the area of forensic toxicology focusing on the screening of drugs of abuse, the usage of alternative matrices for monitoring drug abuse, analysis of chemical warfare agents, determination of doping agents and related substances, natural toxins, environmental poisons, and examination of food produce adulteration. The incorporation of the more user-friendly LC-interfaces, such as atmospheric pressure chemical ionization, and electrospray ionization in the LC- mass spectrometry has increased the popularity of this technique tremendously among scientists of different disciplines. Hyphenated approaches have extremely low constraints regarding the identification and quantification, and offer high reproducibility, with unparalleled potential.

Keywords: Chemical warfare agents, chromatography, drug abuse, forensic toxicology, hazardous substances, high-pressure liquid, mass spectrometry

How to cite this article:
Kumar S, Bogusz MJ. Hyphenated techniques in liquid chromatography and their applications in forensic toxicology: A review. J Forensic Sci Med 2021;7:123-36

How to cite this URL:
Kumar S, Bogusz MJ. Hyphenated techniques in liquid chromatography and their applications in forensic toxicology: A review. J Forensic Sci Med [serial online] 2021 [cited 2022 Aug 12];7:123-36. Available from: https://www.jfsmonline.com/text.asp?2021/7/4/123/334489

  Introduction Top

The unprecedented advent of hyphenated methodology has demonstrated new analytical possibilities for forensic scientists. Hyphenated techniques range from the integration, or linking of chromatographic techniques (e.g., gas chromatography [GC], liquid chromatography [LC], etc.) to spectral techniques (e.g., ultra-violet [UV]-Vis, patent ductus arteriosus (PDA), mass spectrometry [MS], nuclear magnetic resonance [NMR], International Center Of Photography [ICP], etc.), using an effective interface, to maximize the benefits of both these techniques.[1] In hyphenation methodology, chromatography techniques are employed to obtain unblended forms of analytes, from complex chemical compounds, whereas spectral analysis provides standards or the library spectrum required for selective identification information.[1] In 1980, Thomas Hirschfield coined the word “hyphenation,” to describe a possible online combination of separation and spectroscopic analytical methods in a single-run cycle.[2] Hyphenated methods have gained significant popularity in recent years as the primary tool to address complex theoretical challenges.[1]

In the past 20 years, hyphenated analytical methods have evolved exponentially to address complicated analytical challenges. The fusion of separation techniques with spectroscopic technologies is highly effective in the quantitative and qualitative study of suspected substances in complicated matrices.[3] With the aim of acquiring sufficient structural information, for the elucidation of the compounds contained in the rudimentary samples, separation techniques, such as LC, GC, high-performance liquid chromatography (HPLC), or capillary electrophoresis (CE) are coupled with spectroscopic detection technologies, like Fourier-transform infrared spectroscopy, NMR, MS, and PDA, culminated in the development of many hyphenated techniques, such as CE/MS, LC/MS GC/MS, and, LC/NMR.[4] Hyphenation may not necessarily have to be limited to two technologies; the integration of separation and detection technologies may also include multiple separation or detection technologies, such as LC/NMR/MS, LC/MS/MS, LC/PDA/MS, LC/ICP/MS, and so forth.[1] Whenever trace analysis is crucial, and the enrichment of analytes is necessary, online combination with solid-phase extraction (SPE), solid-phase microextraction (SPME), or load value injection (LVI) can be integrated into a more efficient embedded system, e.g., (LVI)-GC-MS or (SPE)-LC-MS.

The goal of this study is to explore the current forensic applications of the hyphenated liquid chromatographic technologies, with particular focus on the screening of drugs of abuse in various matrices, analysis of chemical warfare (CW) agents, determination of doping agents and related substances, natural toxins, environmental poisons and examination of cases of food produce adulteration.

  Identification of Reviewed Studies Top

A comprehensive internet web search of the databases; PubMed/MEDLINE, Science Direct, Scopus, and Google Scholar, for all relevant publications, using the keywords: “hyphenated,” “LC,” “analytic,” “forensic toxicology,” “drugs of abuse,” “trace analysis,” “doping agents,” “dyes,” “natural toxins,” “environmental poisons” and “food adulteration” were performed, using various word combinations. Broad search terms were used to assist the identification of all pertinent original articles, with the last search performed in January 2020. A total of 119 articles that met our search criteria have been included in this review [Figure 2].
Figure 2: Flow diagram for the method used for web search

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  Recent Reviews Published on Hyphenated Chromatographic Methods Top

A number of important reviews have been published from time to time on the applicability of hyphenated chromatographic methods. Tanna et al.(2020) have recently summarized the applications of hyphenated MS techniques in various therapeutic areas showing the increased use of such straightforward approaches to evaluate conformity to medicines.[5] Uliyanchenko (2017), reviewed and discussed the use of hyphenated LC techniques for polymer analysis along with the difficulty for implementing these techniques in laboratories.[6] Sarker and Nahar (2012), outlined the basic operating concepts of various hyphenated techniques employed in natural product research and presented some protocols from the literature as examples of these techniques.[7] Maurer (2020) studied (Hyphenated high-resolution HRMS) approaches and uncovered this methodology that meets the requirements of an all-in-one framework for clinical and forensic toxicology's various applications (such as targeted and nontargeted screening, quantification, drug metabolism, and metabolomics).[8]

This study aims to provide forensic experts with recommendations for selecting a procedure that is appropriate for addressing a particular issue in forensic toxicology. Recent developments in chromatography hyphenation with other instrumental techniques will also be explored, and the reader will be directed to additional comprehensive publications on each subject.

  Review of Applications Top

Drugs of abuse

“Drugs of Abuse” or “Street Drugs” are described as being mind-altering substances, typically taken on nonmedicinal grounds. Substance abuse has been a longstanding problem worldwide for the past 30 years and can lead to physiological and psychological dependence.[9] Testing for substance abuse has now become an integral task in Forensic toxicology, occupational drug testing, anti-doping, and clinical toxicology. Immunoassay methods are used for diagnostic substance abuse testing, and more selective and sensitive procedures are used for validation testing. Analytical techniques like GC or LC, coupled with either MS or MS/MS, are used for confirmation testing, as well as nontargeted drug abuse detection, due to being highly reactive and precise, allowing errorless compound identification.[10] [Table 1] outlines the analytical methods mentioned for the determination of drugs of abuse.
Table 1: Analytical methods described for the determination of drugs of abuse

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Dziadosz et al.,(2018) developed and validated an LC-MS/MS screening method for opiates, cannabinoids, cocaine, amphetamines, benzodiazepines, and methadone in human urine, serum, and postmortem blood as an important replacement to immunoassay-based screening methods. These results indicated that the presented approach satisfies the recommendations for qualitative screening methods and can be effectively used in the study of actual samples.[11] Cox et al.,(2021) validated the QuEChERS extraction and LC/MS/MS system to authentic 34 analytes (liver specimens) including fentanyl, metabolites, and fentanyl analogs.[12] In autopsy cases, Nowak et al.,(2021) employed LC-MS/MS to quantify methadone and its metabolites, 2-Ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP) and electromagnetic dynamic plasticating.[13]

Cailleux et al.(1999) used the precipitation of proteins and fast LC/electrospray ionization (ESI)-MS/MS to quantify cocaine and opiates in plasma, blood, and urine.[14] Successfully, without sample preparation, LC/atmospheric pressure chemical ionization (APCI)-MS/MS was conducted by combining the quantitative analyses of opioids, cocaine, and urine metabolites.[15] Nishikawa et al. presented an LC/APCI-MS system combined with dual SPE for the analysis of cocaine and its metabolites.[16] Bogusz et al.,[17],[18],[19] published research papers on the adaptation of LC/APCI-MS for the analysis of opiate agonists, cocaine, benzodiazepines, amphetamines, and hallucinogens found in various biofluids, such as urine. Hyphenated LC – Tetralogy of Fallot (TOF)-MS is also used to measure cocaine and ecgonine methyl ester in rat plasma.[20] Pharmacokinetic and drug interaction studies,[21] mainly focused on the detection of morphine and its glucuronide, as well as normorphine, in various biofluids, by automated SPE and LC/ESI/MS/MS. In the case of urine drug abuse, an LC/ESI-MS in conjunction with nonautomatic SPE or online SPE were used to identify both amphetamine and heroin metabolites, specifically biomarkers of these substances.[22],[23],[24]

APCI–MS was used to identify cannabinoids in conjunction with super-critical fluid chromatography, while LC-PBI-MS was used to detect the constituents of hashish.[25] Amphetamines (Amphetamine, Methamphetamine, 3,4-Methylenedioxymethamphetamine (MDMA), model-driven architecture, and model-driven architectures) with different sources such as technical support pool, ESI, and APCI sources were identified by LC/MS.[25],[26]

LSD is a highly potent and popular psychoactive drug among substance abusers and is the most difficult to test due to very poor detection of the parent drug in urine.[27] Jang et al., (2015) developed and validated two sensitive and effective methods that rely on LC–MS/MS for the identification of LSD and its metabolite, 2-oxo-3-hydroxy-LSD (O-H-LSD), in urine and hair samples. Hair was analyzed for trace elemental examination using methanol and evaluated with LC–MS/MS. Liquid extraction has been conducted for urine samples.[28] Dolder et al., (2018) demonstrated a responsive LC/MS/MS turbo-flow device for LSD, O-H-LSD, iso-LSD, and nor-LSD quantification and potential detection of LAE, LEO, 2-oxo-LSD, trioxylated LSD, and 13/14 hydro hydrology in human plasma samples.[29] A group of researchers developed a new immunoassay for LSD screening in urine, as well as ESI for urinary LSD confirmation. In addition, this research group used methyesergide as an internal standard for urinary LSD determination in ESI, coupled with SPE. N-methyl–LSD was also detected in real samples.[25] An effective LC/MS/MS system has been established for the identification of nicotine, as well as the key metabolite “cotinine,” in serum samples. LLE, with ethyl acetate, was utilized for serum sample preparation.[30] LC/APCI/MS was also used to determine the nicotine concentration in smokers' and nonsmokers' serums.[25]

In clinical toxicology, LC-MS/MS is gradually being utilized to classify a broad variety of drugs and metabolites in clinical samples. The main benefits of the LCMS/MS method are the simplicity of sample preparation, derivatization not required, the short analysis time, and the simultaneous screening of analytes with greater selectivity and sensitivity. LC-MS/MS has shown ample validity in screening drugs of abuse to bypass prior screening methodologies. The two widely reported atmospheric pressure ionization sources for quantitative or qualitative LC–MS/MS are ESI and APCI.

Novel psychotropic substances

The multitude of new psychoactive substances (NPS) with potentially significant public health concerns has continued to grow in past years. More than 670 such compounds have been confirmed in Europe alone to date and this tends to rise each year as producers and sellers seek to circumvent legislation.[31] For forensic toxicologists, it is a daunting task to identify NPS.[32] Recently, to enhance the detection proficiency towards NPS, many new analytical approaches have been developed including LC–MS/MS or LC–HRMS.[33],[34]

Lendoiro et al.(2017) validated an LC-MS/MS method for the identification of amphetamine-type stimulant, including classical drugs (amphetamine, methamphetamine, MDA, and MDMA), NPS like synthetic cathinones (methylone, mephedrone, methedrone, MDPV, 4-fluoromethcathinone, and (±)-4 fluoromethamphetamine), synthetic piperazines (1-(3-chlorophenyl) piperazine (mCPP) and 3-trifluoromethylphenylpiperazine), and medicines (phenazone and trazodone) that produce mCPP as a metabolite in hair.[35] Likewise, Fagiolo et al., (2018) validated an LC/MS/MS system for detecting 50 NPS in postmortem samples.[36] Adamowicz and Tokarczyk, (2015) have developed a rapid LC/MS/MS screening procedure for identifying 143 NPS in blood.[37] Eckberg et al., (2018) used the LC- quadrupole time-of-flight (QTOF)-MS-based screening approach to construct a reference standard-based spectral library containing HRMS data for over 800 NPS and related compounds.[38] Bade et al. (2019) analyzed 156 wastewater and more than 3,500 forensic samples using LC-HRMS to demonstrate the occurrence of NPS over 5 years in Adelaide, South Australia. There were 24 NPS discovered (six in wastewater, thirteen in forensic post mortem toxicology samples, and five in all). Since these findings indicated the existence of NPS, a systematic technique was used to measure these NPS levels in wastewater.[39] Similarly, Montesano et al., (2016) validated the LC-HRMS system for wide-ranging plasma NPS screening. The approach has been shown to be sufficient for the screening of additional substances, a postrun library matching was performed for each sample with an in-house database containing over 300 NPS and known metabolites.[40] Concheiro et al., (2015) used LC-HRMS for quantifying 8 piperazines, 4 designer amphetamines and 28 synthetic cathinones, and 4 metabolites in the urine.[41] Labuz et al., (2019) showed the efficacy of the LC-FAPA-MS system for semiquantitative detection of legal highs taken by methadone maintenance treatment patients.[42] The hyphenated LC techniques listed for determining NPS are summarized in [Table 2].
Table 2: Analytical methods described for the determination of novel psychotropic substances

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Drugs of abuse in alternative matrix

[Table 3] outlines the applications of hyphenated LC methods for evaluating drugs of abuse in alternative matrices.
Table 3: Hyphenated liquid chromatography techniques described for the determination of drugs of abuse in alternative matrix

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In recent times, it has been found that hair is a possible tool for drug detection in different contexts, such as forensic and occupational testing, as well as monitoring enforcement in drug recovery and drug-facilitated crime (DFC), as any drug and its metabolites deposited in hair, tend to last longer than standard samples.[43] The presence of standard reference material is required for hair testing in substance abuse cases.[44] DFC cases, including different categories of drugs, like benzodiazepines and hypnotics, methadone and buprenorphine can be successfully solved with the help of hair samples.[45]

The (Society of Hair Testing), established in 1995, offers laboratories with proposed best practice guidelines if they actually offer substance testing in hair or intend to do so in future. The standards provide recommendations for sample selection and storage methods, as well as sample processing, pretreatment, and analysis, as well as the use of cut-offs.[46]

Kintz and his colleagues[47] stated that “the sensitivity of LC/MS/MS is highly significant when detecting very small concentrations (pg/mg) of drugs deposited in the hair, in drug-related sexual assault investigations.” Nevertheless, they have introduced a clause in which hair analysis may be considered a complimentary technique for blood and urine testing, rather than an alternative technique. The LC-ESI/MS/MS is especially effective in situations where the concentration of the drug is very small, as only one single therapeutic dose accumulates in the hair, such as benzodiazepines and benzodiazepine-like hypnotics.[48]

In a randomized trial to examine the detection window for lorazepam in semen, hair, and oral fluid (OF), the value of LC/MS/MS was discovered due to simultaneous examination of 26 benzodiazepines, zolpidem, and zopiclone in blood, hair, and urine.[49] The LC-ESI-MS/MS system has also been introduced to genuine samples of both forensic and clinical cases, such as the examination of a woman's hair who had claimed to have been drugged and sexually assaulted for many years shows the utility of hair, in the sense of chronic drug use. Hair samples were screened for ethyl glucuronide which is considered a marker of chronic alcohol use. LC/MS/MS and GC/MS are used for such determination.[50] In an unintentional overdose case, Larabi et al.(2020) demonstrated the effectiveness of hair scanning for fentanyls using LC–MS/MS.[51] Musile et al., (2020) employed UHPLC-Ion Trap-MS in routine contexts for broad range of screening of hair samples for drugs of abuse.[52]

Oral fluids

OF can be exploited as a substitute matrix for the detection of substance abuse in occupational testing, clinical toxicological examinations, criminal enforcement cases, and DUI-cases.[53] The OF matrix provides a fairly clean and secure way for sampling, thereby reducing the possibility of sample adulteration. Protein precipitation (PPT) followed by LC/APCI-MS/MS was used for the determination and quantification of opiates, cocaine, and its metabolites found in OF.[54] Vindenes et al., (2011) utilized LC/MS/MS to detect different drugs and their metabolites (n = 32), collected from the OF of real-life patients undergoing a drug treatment program, and the findings were simultaneously compared to urine samples taken from the same patients.[55] In addition, Welch et al., (2003) identified a procedure for detecting methadone and other addictive substances and their metabolites in hair.[56] PPT with acetonitrile, accompanied by LC/MS/MS analysis was utilized in this procedure and was particularly effective for evaluating methadone concentrations in pregnant women. The Food and Drug Administration has approved a screening tool used for large-scale workplace drug monitoring in the United States, and it was under review, in a new EU-US roadside trial to identify driving under the influence of drugs (DUID.).[57] Spanish researchers used liquid-liquid extraction (LLE) with hexane for the processing of OF specimens for analysis.[58] Pascual-Caro et al., 2020 have developed a rapid LC/MS/MS procedure for determining synthetic cathinones in OF with strong potential for the determination of other drugs in OF.[59]


Drug misuse is a significant concern during pregnancy and is linked to prenatal problems and elevated incidences of infant morbidity and mortality.[60] Many birth defects are believed to be due to prenatal drug exposure.[61] Urine drug testing has historically achieved the identification of drug exposure in the uterus. This, however, only indicates maternal drug usage within the last 34 days and a mother's abstinence several days before delivery may produce a negative outcome. Alternative matrices, like neonatal meconium and hair, can be used to monitor exposure, including earlier gestational noninvasive collection and detection.

Meconium is the new-born's first stool, usually in the immediate days after birth. Meconium production begins between 12 and 16 weeks in fetal development and deposits continue in the intestine of the fetus until birth. The presence of this substance can considerably delay the drug detection period that is up to about the last 20 weeks of pregnancy. Meconium sample testing is a time-consuming process. Compared to liquid samples, meconium is a sticky, semi-solid, tar-like excretory product and should be weighed before extraction. Goggin et al., (2019) described a process by which ceramic homogenizers were used before the extraction of salt-assisted LLE and LC/MS/MS to analyze cocaine in meconium along with its metabolites and amphetamines.[62]

Ristimaa et al., (2010) analyzed abused drugs in meconium within a routine examination, utilizing LC/MS/MS and TOF-MS technology.[63] Jensen et al., (2019) developed an LC/MS/MS system for identifying and quantifying 4 cannabinoid alkaloids in neonatal matrices.[64] Choo et al., (2005)[65] employed LC-APCI-MS/MS for drug testing in meconium. Gray et al., (2009) also used meconium for the detection of opiates, cocaine, and amphetamine with the presence of their respective metabolites. In both instances, the samples were prepared using SPE, and the experiment was carried out utilizing LC–MS/MS. LC/MS/MS quantified the drugs in meconium samples in nanogram per gram range.[66] In another study, LC/MS was used for quantifying methadone, cocaine, 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine, benzoylecgonine, morphine, 6-acetylmorphine, and codeine in the human placenta.[67] This hyphenated technique can be employed to detect traces of methadone in meconium, which may have been ingested continuously by the mother of the fetus, throughout pregnancy.

Postmortem alternative specimens

It is clear that alternative specimens (e.g., muscle tissue rather than blood) have proven to be highly advantageous in the detection of antemortem substance addiction, as well as being valid in postmortem procedures.[68] Toxicological analyses of conventional postmortem specimens pose problems, in the case of decomposition and putrefaction of specimens. The absence of any suitable biological substance or fluid may be replaced by entomological evidence and is often utilized to confirm the existence of drugs in the organism. Though insect tissues have proven to provide reliable samples, it is certainly not possible to claim that drugs were the cause of death.[69] Wood et al.,(2006) developed an LC/MS/MS system for quantifying benzodiazepines in larvae and puparia of Calliphora vicina.[70] The efficacy of this system has been verified by the study of larvae and puparia, reared on media that were spiked with nordiazepam, at a concentration of 1 μg/g. Oxazepam present in larvae or pupae, as a compound or metabolite. Different stages of the larva can be utilized to estimate the postmortem interval. During such instances, it has been found that the presence of drugs has an effect on insect development; as a consequence, such disruptions could have serious implications on the precision of estimating the time elapsed since death.

Pelander et al., (2010) analyzed the usefulness of vitreous humor (VH) as an additional matrix for drug screening with LC/TOF/MS in postmortem investigations, by comparing results in VH and urine, in 50 autopsy cases. The comparative study revealed that VH is suitable for qualitative screening analysis, but its generalizability was not as effective in urine, due to less frequent metabolite detection. In total, 45 parent compounds and 24 metabolites were detected in VH samples; for urine, respectively. The mean and median cut-off values were respectively 0.072 and 0.023 mg/L which was acceptable for case handling.[71]

Chemical warfare agents

CW is perhaps one of mankind's deadliest weapons of mass destruction. Usually, they are extremely hazardous and highly lethal synthetic compounds which may be dispersed as liquid (as well as aerosols), gas, solid, or adsorbed on to particles, to become powder.[72] Hundreds of hazardous chemicals are recognized, but very few are deemed to be CW agents based on their characteristics.[73] This group includes simple molecules like chlorine and complex structures like ricin. CW agents include nerve agents, incapacitating agents, vesicants, lung-damaging agents, blood agents, riot-control agents, and a variety of toxins.[74] The nerve agent is one of the most lethal of CW agents and is likely to be used by terrorists against civilians in an attack. The nerve agents generally contain organophosphorus compounds which are recognized as among the most toxic of substances.[75]

The possibility of using CW agents in domestic terrorist activities has been displayed for the very first time in these cases.[76] Investigations have subsequently confirmed that Tabun, soman (GD), ethyl ([2-[bis[propan-2-yl] amino] ethyl] sulfanyl)(methyl) phosphinate (VX), and Botulinum toxin have also been manufactured by the sect.[77] Therefore, only limited numbers of people can utilize CW agents to cause vast and pervasive human misery without warning. Biological samples are also tested to confirm exposure, in order to identify CW agents, along with their metabolites or degradation products. Nerve agents, also referred to as nerve gases, are volatile compounds and therefore examination using GC/MS can be deemed as an easy alternative. Despite this, such agents are rapidly hydrolyzed to alkyl phosphonates in an aqueous environment, which are then subsequently hydrolyzed into methyl phosphonate. LC/MS is gradually being utilized for such highly polar, low-molecular-weight compounds, although at the same time taking advantage over GC/MS, of reduced sample handling without derivatization.

Another common technique that blends the separating power of LC with the highly selective and sensitive mass analysis capability of MS/MS. Hamelin et al.,(2014) validated the SPE-LC–MS/MS system, for the study of short-lived metabolites of many CW agents, like Russian VX (VR), sarin (GB), cyclosarin (GF), GD, and VX in serum.[78]

Smith (2004) utilized normal-phase LC in conjunction with MS detection and validated an LC–APCI-MS system for VX.[79] Some researchers utilized LC–MS to identify the metabolites of (sulfur mustard), i.e., the β-lyase metabolites in urine samples.[79] D'Agostino et al., (2005) developed and validated an LC–ESI-MS/MS system that enables the detection of a munitions grade sample of tabun, GB, GD, GF, and the nerve-stimulating agent triethyl phosphate (TEP) on man-made fibers.[80] In recent times, they have experimented by completely skipping sample preparation and enabling the direct examination of TEP collected on SPME fibers.

Ricin, a toxic glycoprotein produced from castor bean plant seeds (Ricinus communis), is among the most potent toxins known to humankind.[81] If the purified toxin is ingested or inhaled, a single castor bean contains enough ricin to kill 1000 people. Ricin also has the unusual status of being the only protein identified under the Chemical Weapons Convention and is of forensic significance because of the propensity for extremist use, as well as an agent of assassination.[82] Utilizing nominal mass analysis, absolute structural elucidation of the intact protein is not feasible, owing to the large molecular weight of about 64–65 kDa. Nonetheless, some researchers used enzymatic digestion to transform protein to intermediate molecular weight peptides accompanied by LC–QTOF/MS/MS. Several methods have been employed to classify purified ricin from many specific types of R. communis, and even too from crude extracts of castor beans.[83]

Feldberg et al., (2021) devised an LC–MS/MS method for ricin identification in clinical samples, which has the potential to be used to identify other lectin toxins.[84] Kanamori-Kataoka et al., (2011) developed a fast and reliable method for evaluating ricin by nano LC/MS/MS after extraction using lactose-immobilized monolithic silica spin column. Due to the extremely high detection sensitivity in MS and the collection of accurate data from separate protein digests, nano-LC/MS/MS was chosen.[85]

Ma et al., (2014) validated an LC-ESI-MS/MS technique for identifying and quantifying residual ricin in poisoned rats' serum. These findings indicate that residual ricin in poisoned rats serum can be detected up to 10 ng/mL even after 12 h exposure.[86] Liang et al., (2020) used trypsin/Glu-C tandem digestion in combination with UHPLC-MS/MS to detect ricin in beverages. This study offers a realistic analytical approach for accurately quantifying ricin in dynamic matrices, as well as a new technique for identifying protein toxins in public safety.[87]

Direct mass spectrometric analysis of CW agent degradation products has recently been detected by atmospheric pressure ionization (ESI and APCI) techniques. LC-ESI-MS/MS methods have recently been employed for the study of nerve agents and also used to supplement certain GC-MS methods for the analysis of infected aqueous extracts or samples. The hyphenated LC techniques stated for evaluating CW agents were summarized in [Table 4].
Table 4: Hyphenated liquid chromatography techniques described for the analysis of chemical warfare agents

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Doping agents and related compounds

The usage and misuse of performance-enhancers have been a concern in competitive games since antiquity. About 30% of players competing in the 2011 World Championships in Athletics confirmed that they had used banned drugs during their careers. In accordance with a survey conducted by the World Anti-Doping Agency, 44% of players still use them and just 0.5% of those tested were captured.[88] The emergence of different synthetic steroids, stimulants, and peptide hormones has posed an analytical challenge. Several contributions to the determination of steroid compounds were developed. The hyphenated LC techniques listed for evaluating doping agents and related compounds were summarized in [Table 5].
Table 5: Analytical methods described for the analysis of doping agents and related compounds in biological matrices

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Nowadays, sensitive LC/MS systems are readily accessible for nearly all types of performance-enhancing drugs. The first use of LC/MS in anti-doping analysis was the identification of highly polar and thermally labile diuretics.[95] Since soft ionization interfaces like ESI or APCI are commercially viable, the LC/MS/MS technique has pioneered detection assays employed in doping control research. The LC/MS/MS system can detect anabolic androgenic steroids, β2-agonists, hormone antagonists and modulators, glucocorticosteroids, and beta-blockers.[96] Most classes of diuretics are identified in urine using either LC/ESI/MS/MS or LC/APCI/MS/MS at reasonable LOD with high specificity.[97],[98],[99]

Thevis et al., (2009) studied Trenbolone (17 β-hydroxy-estra-4, 9, 11-trien-3-one) and its derivatives, like 17Ш-methyltrenbolone, utilizing LC/MS/MS.[89] Tang et al.(2001) have validated the LC/APCI/MS/MS system in equine urine for the simultaneous screening of 21 synthetic corticosteroids, deoxycortone, and hydrocortisone.[90] Jeon et al., (2011) have set up a robust and efficient LC/MS/MS system with ESI for the identification and validation 44 exogenous steroids in urine, with the LOD for the screening and confirmation methods being 0.1–10 and 0.2–10 ng/mL, respectively. This approach has been used extensively for urinalysis acquired from alleged abusers of anabolic steroids.[91] LC/ESI+‒MS/MS can be used to detect 4-Methyl-2-hexaneamine doping agent in urine.[92] Mazzarino et al.(2010) used a single chromatographic approach to screen several classes of substances in 11 min. The efficacy of this technique was assessed using LC/ESI/MS/MS in the + ve mode on 20 blank urine samples spiked with 45 forbidden compounds in sport, comprising 16 glucocorticoids and 9 stimulants. In urine, all analytes were easily distinguishable, with LOD varying from 5 to 350 ng/mL. All of the compounds of interest, including synthetic and endogenous glucocorticoids, were isolated with identical retention periods and fragmentation patterns.[94]

Thomas et al., (2014) developed an LC-IM-MS technique for determining insulin and its synthetic as well as animal analogs insulin aspart, glargine, glulisine, lispro, detemir, porcine, and bovine insulin.

LC–MS/MS can be used to test trace compounds with high selectivity and sensitivity, and it can be employed to quantify and identify forbidden substances in biological samples. Due to low endogenous quantities, the presence of several related molecules, and matrix interferences, quantitative study of a wide variety of doping agents in human serum is difficult. A considerable amount of sample preparation is needed for an SRM-based analysis. The difficulty in achieving the optimal sensitivity and dynamic range is exacerbated by water losses during ionization in both ESI and APCI sources, as well as extensive fragmentation in the collision cell.

Natural toxins

Natural toxins have been used since ancient times to create poison arms for carrying out the first types of biological warfare and CW.[100] Natural toxins are harmful, organic compounds, of natural origin. Several of these toxins include: Glycoalcaloids, Mycotoxins, Aflatoxin A1, A2, B1, B2, Aflatoxin M1, Patulin, Ochratoxins, Lectins, Cyanogenic glucosides, Amygdalin, Tetradotoxins, as well as Fish and algae toxins. Owing to the loss of annual revenue due to contamination, toxins are toxic to both humans and livestock (most are documented carcinogens), and are ever present during several food processing phases, which is of major concern to global economies. Toxic constituents need to be accurately registered to establish a prefect repository for use in Forensic analysis and the identification of particular causal agents.

Ogawa et al.(2020) created a forensic toxicological archive for the analysis of 56 natural toxic compounds employing LC/QTOF/MS/MS. The usefulness of the archive was evidenced by the identification of 4 plant toxins in blood samples, taken from postmortems.[101] Choe et al., (2018) showed that even without reference materials, LC-Q-TOF-MS/MS analysis was an effective method for mushroom intoxication cases.[102]

Puntscher et al., (2018) documented the LC/MS/MS system for the simultaneous identification of 17 Alternaria toxins in three food/feed matrices, often contaminated with these pervasive natural toxins, including sunflower seed oil, tomato sauce, and wheat flour.[103] Bisseli and Hummert, (2005) have developed an effective, sensitive and selective system for the systematic analysis of various Fusarium mycotoxins (Type A and B trichothecenes, zearalenone) in cereal samples, utilizing LC-ESI-MS/MS.[104]

Environmental poisons

LC/MS has been the most commonly employed analytical method for pesticide detection. Several studies in the last decade have reported on pesticide analysis and products by LC/MS.[105],[106] LC/APCI/MS with positive ion mode is found to be sensitive to certain phenylureas.[107] Goto et al., (2006) established a new method of analysis for 9, N-methyl carbamate (NMC) insecticides in vegetables and fruits utilizing ESI-LC-MS/MS.[108] Alkyl phosphates and alkylthiophosphates studied by LC-(ESI-)-MS/MS.[109],[110] Shin et al., (2018) outlined a simultaneous screening method for evaluating 379 pesticides in serum samples utilizing LC/MS/MS. The LC-ESI-MS/MS system with negative/positive ion-switching ionization modes was introduced, and scheduled multiple-reaction monitoring for each target compound were carried out.[111] For 94.5% of the total pesticides, the limit of quantitation (LOQ) was 10 ng/mL, and the calibration coefficients for 93.9% of the pesticides were ≥0.990. Pyrethroid metabolites are analyzed frequently using LC/MS/MS.[111]

Food additives

The science involved in criminal forensics is often used to investigate issues with food safety and quality.[112] Food adulteration may have significant implications on peoples' health and may also impede business development, by the loss of consumer trust. Additives like transglutaminase, typically referred to as meat glue, are approved for use, to bind together meat trimmings and off-cuts; but similar meat bindings have also been stated in instances of fraud, where criminals have attempted to include them to deliberately increase the weight of meat material shown on labels.[113],[114] Many unethical activities also include the usage of blood plasma fibrin protein, plus thrombin, to bind pieces of meat, that could include combining blood thrombin fibrin protein from various species (for example bovine, porcine). Specialists in the field are utilizing more selective and sensitive methods, as well as analytical techniques, to measure and monitor food adulterants, which are both easy and cost-efficient.

In regard to this, Grundy et al., (2007, 2008) developed and validated LC-ESI-MS(/MS) and LC/MS/MS capable of distinguishing fibrin clots exploited in binding procedures and may indicate the origin of mixed species in blood.[113],[114] LC/MS or LC/UV is used in conjunction with quantitative NMR spectroscopy (qNMR) to confirm adulteration cases in other food products, like monofloral-honey, owing to the existence of unusual and very sensitive biomarkers in the nectar of plant species used by the bees.[115] The LC/MS/MS system was proposed to validate the potential existence of nuts (as well as peanut) allergens in chili peppers.[116] Coupled with the chemometric examination, LC-MS can supplement current methods for detecting adulterated juices.[117]

Marchesini et al., (2007) coupled a fast dual biosensor immunoassay (BIA) with high-resolution LC/ESI-TOF-MS to screen Fluoroquinolones (FQs) and aid in identifying known and unknown compounds in noncompliant chicken muscle samples. This method also detects unknown chemicals of identical structure that exhibit activity in the dual BIA.[118] Yusop et al., (2021) used LC-HRMS to examine ED drugs and analogues in (powdered drink mix), jelly, hard candy, honey, and sugar-coated chewing gum with a LOD of 10–70 ng/mL and a LOQ of 80 ng/mL, demonstrating the excellence of the techniques in terms of precision and linearity.[119]

Because of the large number of bioactive compounds and relative derived-degradation products that can be analytically controlled by a single technique, LC–HRMS is now the most widely used approach applied to a broad range of food products.

  Conclusions Top

The application of the hyphenated analytical method in forensic toxicology research has grown significantly in recent years. The popularity of the LC-MS technique, incorporating more user-friendly LC-interfaces, for example, APCI and ESI have gone up tremendously. LC/MS/MS techniques utilizing serum, plasma, and urine samples are the most common. Matrix effects have the potential to reduce the efficiency of LC/MS/MS approaches and must therefore be properly evaluated during method validation. LC-HRMS approaches and alternative biosample matrices such as hair, saliva, and blood microsamples have received significant attention in the last few years. Due to its high instrumental sophistication and cost, HRMS techniques, like Orbitrap, (Fourier-transform ion cyclotron resonance), and ToF have generally been restricted to use in well-equipped labs.

Hyphenated methods address several applications and are becoming increasingly relevant and noticeable in almost every area of science. Hyphenated approaches have extremely low constraints when it comes to identification and quantification, and offer high reproducibility, with unparalleled potential. The sophistication of the instrumentation and the complexity of the process are the main barriers to the standardization of hyphenated techniques.

Financial support and sponsorship


Conflicts of interest

There are no conflicts of interest.

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  [Figure 1], [Figure 2]

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5]


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