Environmental Toxicology

Forensic Science International: Reports 5 (2022) 100275

Available online 26 May 2022 2665-9107/© 2022 The Author. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by- nc-nd/4.0/).

Chronology of preceding medico-legal practices with Reference to post-mortem forensic toxicology

Rajvinder Singh 1

Department of Forensic Science, Maharshi Dayanand University, Rohtak, Haryana 124001, India

A B S T R A C T

Exercising an accurately effective extraction method on the post-mortem samples is one of the integral as well as imperative parts of a successful forensic toxico- logical analysis. Scientific progress has witnessed quite a few efficient attempts made to effectively extract poisons from post-mortem samples. Lack of expert knowledge or unwanted procedural error, even the minor while adopting these methods can affect the scientific conclusions and the legal verdict as well. A sys- tematically appropriate means of extraction is evenly dependent on the nature of poisons typically grouped in the past for medico-legal purposes; therefore, it significantly necessitates the expert acquaintance with the classification of poisons in depth. Deciphering such aged important information isn’t an easy task due to a lack of substantial forensic appraisal; therefore, the present review articleattempts to augment essential knowledge on the classification and extraction of poison sought for forensic rationale. This article has also integrated biographies of associated pioneers with the purpose to salute their commendable work and inspire interested readers. This compendium can adjoin the modern toxicologists with the origin and understanding of convectional toxicological practices aiding the legal system.

1. Introduction & background

Over time, the legal scrutiny of deaths with poisoning followed stages of post-mortem findings, extraction/isolation of poison, result interpretation, and expert testimony in the court of law [1]. Forensic toxicology is a hybrid of analytic chemistry and elementary toxicolog- ical principles and is primarily practical for the medico-legal aspects of human poisoning [2]. Of the various specialized branches, forensic toxicology is one of the oldest and pillar subsets of forensic chemical sciences. Forensic toxicology has its traditional as well as contemporary linkage with the subject of forensic medicine or medical jurisprudence [3]. The post-mortem examination of the deceased under the observa- tion of a qualified medical examiner expands to take out verities of biological samples, especially tissue and body fluids to expose poisonous substance/s responsible for the death. The expert probe then shifts from the mortuary room to a forensic science laboratory to search for conclusive scientific evidence. Expert toxicologists apply their skill and experience to identify poisons, if any, responsible for the death, and finally present all related and essential facts of scientific analysis in the court of law accountable in the final legal verdict [2,4,5]. The analysis commences with extraction procedure following qualitative and quan- titative scrutiny of analytes adopting accurate and strategic methodol- ogies cased with quality controls. Analytical responsibility entirely rests with a forensic toxicologist. Any success or failure of scientific

examination is fully bound to the receipt of correct and sufficient au- topsy specimen, application of suitable extraction procedure, and so- phisticated methods of analysis.

Term ‘extraction’ here is a scientific operation of withdrawing an agent or substance from a solid or liquid biological matrix with the help of selected chemical salts and solvents, etc. Many renowned analytical chemists and toxicologists desirably but systematically classified poisonous substances way back when the development of the extraction procedures was taking place while the medico-legal subject was advancing to deal with poisoning cases. An effective and accurate sci- entific recovery of such poisons in the biological samples forms an imperatively decisive factor in the final legal verdict. A scientist without acquiring a considerably sound basic scientific knowledge of poisons and their extraction from biological specimens might not become an expert in forensic toxicology. Consequently, the current paper has narrated a compendium of a few forensically relevant systems of clas- sifying poisons and their established extraction methods for medico- legal and forensic excellence.

Toxicology has been an integral part of cultures from ancient times ever since the disclosure of several famous cases of criminal poisoning in the progress of legal toxicology. The eighteenth, as well as nineteenth century, witnessed huge temptation of poisoning cases that paved the way for the beginning of the modern era of toxicological studies. In the nineteenth century, the easy availability of noxious poisons raised

E-mail address: [email protected]. 1 https://orcid.org/0000-0003-4209-7659

Contents lists available at ScienceDirect

Forensic Science International: Reports

journal homepage: www.sciencedirect.com/journal/forensic-science-international-reports

https://doi.org/10.1016/j.fsir.2022.100275 Received 16 March 2022; Accepted 16 May 2022

Forensic Science International: Reports 5 (2022) 100275

2

prevalent communal anxiety about “poison panic” (murder by poison) in Europe and the United States. These types of crime played a crucial role in developing the convectional medico-legal toxicology, obviously with the experimental linkages of distinguished chemists cum expert wit- nesses including J. Marsh, M.J.B. Orfila, A.S. Taylor, J.S. Stas, and some others [6]. These eminent scholars massively contributed to the body of forensic toxicology with their skill and knowledge. Later in the twentieth century, another great A.S. Curry significantly added his research to raise emergency and post-mortem toxicological analysis status. The contribution of the Indian eminent, Jaising Prabhudas Modi (J.P. Modi), besides deserves a commendable say during the early twentieth century.

This era is marked by the advancement of toxicology with instru- mental analysis, and the level of cellular molecules and biochemical moiety. Scientific inputs of these pioneers helped grow up forensic toxicology and the classification systems of poisons and their extraction directly or indirectly. It is now well understood that the acquaintance of types of poisons and their extraction from the autopsied biological specimens are two primary mandates of forensic toxicological analysis. These two aspects have a direct association and impact in forensic toxicology for better strategizing the analysis in poisoning cases. This article has traced the history of the classification of poisons and con- ventional extraction methods along with biographies of the pioneers who were allied to these two sides. Reviews and discussion are highly restricted to medico-legal and forensic aspects.

1.1. Relevant history of classifying poisons

As far as legal toxicology is concerned, no chemical substance can be left out of suspicion of identification. Thus, forensic toxicologists had established a systemized standard approach when the nature of any poison is anonymous [2]. Definition of poison needs full understanding before knowing the classification of poisons. The word ‘Poison’ has been defined several times since its origin. Definitions of poison are a little complex, but one stated by Philippus Aureolus Theophrastus Bombastus von Hohenheim or Paracelsus (1493–1541) was the correct and broadly accepted. Paracelsus for the very first time, defined a poison as “all substances are poisons: there is none which is not a poison, the right dose differentiates a poison and a remedy” [7,8]. Analytes in the form of toxicants/poisons must be thoroughly understood and classified before extracting them from post-mortem samples. After quite a while, the origin was squeezed, and occasionally, the bases of classification shifted to their methods of isolation/extraction in medico-legal toxicology.

1.2. Classifications of poisons according to the mode of action and effects

Pedanius Dioscorides, also known as Pedanius Dioskourides (40–90 CE) was a Greek clinician and one of the first pioneers who made a convincing attempt to classify poisons into the plant, animal, and min- eral groups accompanied by their descriptions and drawings [2,9]. Another highly imperative classification of poisons was given by M.J.B. Orfilia (1818) in his memoir ‘Traité des poisons, tirés des reégnesminéralvégétal et animal; outoxicologiegénérale, considérée sous les rapports de la physiologie, de la pathologie et de la médecinelégale’ [10]. He divided poisons into (i) corrosives, (ii) astringents, (iii) acids, (iv) stu- pefying or narcotics, (v) acrids, and (vi) septics or putreficants, justifying the legal jargon [11]. Another forensically vital reference ‘Poisons, concerning Medical Jurisprudence and Medicine’ reported that the mid-nineteenth century notified the classification of poisons according to their Kingdoms of origin [12]. In the same way, Chapuis E. (1873) in ‘Elements de Toxicologie’ also recommended the categorization of toxic substances based on their origin or nature [13,14]. As a result, poisons were classified into mineral, vegetable, and animal poisons, but the utility of such a classification was not helpful enough in acquiring ab- solute knowledge, even subordinating to a physiological category. Taylor (1848) believed that all such classifications are inevitably arbi- trary to some extent but preference depends upon the interest of the

analyst. Therefore, keeping in the interest of toxicologists, he divided (modifying Orfila’s classification) poisons according to their mode of action on the target system into (i) Irritants (violent vomiting and purging symptoms with intense abdomen pain), (ii) Narcotics (affect central nervous system leading to suffering from vertigo, paralysis, coma, etc), and (iii) Narcotico-Irritants (compound action and chiefly derived from the vegetable kingdom). Poisons of organic and inorganic origins were also known in ancient India. An Indian standard system of classification of poisons as given by J.P. Modi in his book ‘Modi: Medical Jurisprudence and Toxicology’ is a highly followed reference by the Indian medico-legal and forensic professionals. This system of classifi- cation followed the mode of chief symptoms and divided poisons into 1) Corrosive, 2) Irritants (organic and inorganic), and 3) Systemic, affecting CNS, cardiovascular and respiratory systems. He also added that any particular poisons could have multiple signs and symptoms in multiple body organs; therefore, it is difficult to place them in the given classi- fication for the simplicity of this systematic [15]. All relevant details have been shown in Fig. 1.

1.3. Classifications according to extraction methods of poisons from the human body

A scientific concept systemizes the execution of an associated extraction method for separating similar types of poisons or groups of poisons from the biological matrix. The use of appropriate extraction methods in forensic toxicology depends upon the physical and chemical nature of poisons; therefore, broad types of poisons are categorized into various groups involving volatile, gases, metallic, drugs, anions, pesti- cides, miscellaneous, etc. In the abstract, the understanding of classi- fying poisons becomes logically essential and imperative before applying a suitable method of their extraction from the biological samples. Chemists started strategizing isolation of metallic and alkaloid poisons from varieties of media long back in the eighteenth century [2]. Some highly credible and significant contributions to classifying poisons as per their methods of extractions, particularly from the medico-legal point of view have been given in ‘Poisons their isolation and identifi- cation’ [16], Clarke’s analytical forensic toxicology [7,17], and ‘Casarett and Doull’s Toxicology: The Basic Science of Poisons’ [2,18,19]. History reveals a good piece of related work published by Bamford [16]. From the analyst’s point of view, the most helpful method (not mutually exclusive) of classifying poisons was based on schemes for isolating poisons (seven groups) from the biological samples. This classification included seven groups i.e. (i) volatile poisons (alcohols, chloroform hy- drocyanic acids, etc) isolated by distillation; (ii) alkalis, acids, and irritant salts of alkali metals extractable with water; (iii) common metallic poisons (mercury, arsenic, antimony, and bismuth) deposited on copper foil when heated with HCI; (iv) other poisonous metals; (v) non-basic organic poisons soluble in ethyl alcohol, and somewhat soluble in water or dilute alkalis, extractable in acidified solutions by shaking with ether, chlo- roform or other immiscible solvents; (vi) basic organic poisons usually extractable with Stas-Otto process; and (vii) other substances (e,g. Abrin, Ricin, and other many vegetative materials) isolated with dialysis considered to be a special method. He also mentioned that these groups were deemed not mutually exclusive because a few substances could have been put in more than two groups and substitution of extraction method as well. Another important submission was the exclusion of the second and the seventh group out of the seven classes of poisons. The reason quoted for group second was the inevitable detection of acids, alkalis, and irritant salts of alkali metals during preliminary medical and pathological examinations. The substances of the seventh group were thought to have not been fitting into any general scheme but rather required different special methods for their detection [16].

‘Casarett and Doull’s Toxicology- The Basic Science of Poisons’, a highly valuable book series that nurtures toxicology concepts of the students and scientists from interdisciplinary disciplines. This book is a prestigious reference in providing the basic and core information on

R. Singh

Forensic Science International: Reports 5 (2022) 100275

3

several facets of forensic toxicology through its chapter ‘Analytic/ Forensic Toxicology’ written by Professor Alphonse Poklis from Virginia Commonwealth University, Virginia. The term ‘toxicant’ in this book means any toxic substance produced by or is a by-product of anthro- pogenic (human-made) activities’. According ‘toxicants’ have been categorized into seven groups i.e. i) gases (e.g. carbon monoxide); ii) volatile substances (e.g. alcohols and other chemical liquid types); iii) corrosive agents (e.g. mineral acids and bases); iv) metals (e.g. ions of mercury, and lead, etc.); v) toxic anions and non-metals (e.g. phos- phorus, etc.); vi) non-volatile organic (drugs, pesticides, and industrial compounds, etc); and vii) miscellaneous substances (e.g. proteins mix- tures of venoms, etc.). This system of classification based on the origin or nature of the toxic agents was proposed by Chapuis (1873) in ‘Elements de Toxicologie’ [2].

Likewise, an acquaintance with a highly reasonable reference i.e. ‘Clarke’s analytical forensic toxicology, is a must for getting close to the classification of poisons and their extraction from a forensic perspective. The contents embedded in this book’s editions have properly addressed the forensic toxicological issues and technically enhance the knowledge of students and researchers studying forensic toxicology and analytical chemistry. Clarke’s Analytical Forensic Toxicology7 adapted from Moffat et al. [20] systemized poisons into five major groups i) gaseous and volatile substances (isolation by distillation or GC-HS), ii) organic non-volatile (by solvent extraction), iii) metallic poisons (by ashing/wet oxidation/ enzymatic hydrolysis of the tissue), iv) anions (by dialysis),

and v) miscellaneous poisons (immunoassays/ ion-exchange colum- ns/freeze drying/continuous extraction with a polar solvent). But keeping in view the quantum of substances and availability of alterna- tive methods of extraction in the group (i) and (ii), the gases were separated from the group (i) and similarly, group (ii) gave way to another group i.e. pesticides. Now there were seven groups in total classifying poisonous substances as per their methods of extraction in forensic science, as shown in Fig. 2.

1.4. Pioneers and their established extraction methods

A literature survey revealed few highly spoken chemists-cum ana- lysts who dedicatedly gave new meanings to medico/chemico-legal toxicology by developing methods to extract poisons of forensic signif- icance. Many of these fundamental extraction procedures for poisons were sound enough in the legal verdicts. A few complicated legal trials, including the Bocarmé murder (1850), suspecting alkaloidal poisoning put some medical experts in the battle of scientific debate in the court of law during the early period [21]. The nineteenth century, in particular, was the bystander of two milestones i.e. the ‘Marsh test’ and ‘Stas-Otto’ extraction procedure in the legal toxicology. Literature depicts that arsenic and nicotine-like alkaloidal poisons were vastly studied in the past, especially from a medico-legal point of view. Followings are the revisited biographies of few pioneers of this field:-

Fig. 1. Classification of poisons according to the mode of action and chief symptoms [3,15].

R. Singh

Forensic Science International: Reports 5 (2022) 100275

4

1.4.1. James Marsh (1794–1846) The developer of the ‘Marsh test’, James Marsh, a British chemist

analyst was born on the 2nd of September 1794 in Woolwich district in the southeast (then just outside) London. Marsh worked on several burning medico-legal issues of that time, including analysis of arsenic poisoning. Exercising his scientific skill, Marsh cracked the anonymity of arsenic poisoning by developing its extraction method within biological samples. He revamped ordinary conventional testing based on precipi- tation and the reduction method to develop a simple and more reliable the ‘Marsh test’ for detecting trace amounts of arsenic in foodstuff and some other materials [22]. Marsh died on the 21st of June 1846 in the Arsenal, London. There were a few crucial reasons behind vastly studied metallic and alkaloidal substances in the legal cases.

1.4.2. Arsenic as a motive poison Use and nomenclature of arsenic is found in several old civilizations

by exploring various folds of literature dictated by Aristotle (350 B.C.) Theophrastus (300 B.C.), and Dioscorides (50 B.C.), Paracelsus [23–26] including from India and the far East during somewhere in 2000 B.C [27–30]. Arsenic poisoning in the past posed a massive problem due to the resemblance of its signs and symptoms with cholera and pneumonia-like diseases [31] and its impracticable detection in the dead body [32]. Therefore, It was believed that arsenic was more frequently used for criminal purposes than any other poisons [33].

1.4.3. Background of the ‘Marsh test’ (1836) Arsenic was once a considerable homicide poison of medico-legal

importance due to its impracticable detection in the dead body. The literature revealed several chemical analyses for the detection of arsenic including Hahnemann’s Test [34] with unsatisfactory, false positive, and time-consuming results [35,36]. An important case came in 1833 when the law enforcement agency produced young John Bodle for a legal trial at Maidstone court in England. He was accused of murdering his grandfather by serving his coffee with arsenic-based rat poison. Marsh examined the case in the capacity of a chemico-legal expert [22]. Marsh applied a conventional chemical test (Hahnemann’s test) to detect arsenic in the case exhibits but the jury couldn’t infer the unclear scientific results. The accused was acquitted due to a lack of substantial scientific evidence. Marsh then realized the limitation of traditional tests but he was capable enough to resolve this issue with his practical and theoretical skills of understanding poisons and their effects [37]. Conquering his disappointment, a few years later, Marsh announced a

new, sensitive, and highly dependable test that soon turned out to be the “chief terror of poisoners,” exposing arsenic-related criminal poisoning and reducing the quantum of using arsenic for murdering [38,39].

Marsh revisited Scheele’s old method to formulate his new chemical test for the detection of arsenic poison in the biological samples [7]. It is evident from the literature that C.W. Scheele (1775) found that when zinc (Zn) is added to arsenic oxide (As2O3) dissolved in dilute nitric acid, a colorless gas with garlic stink gas is evolved along with hydrogen known as ‘arseniuretted hydrogen’ or arsine (AsH3) as per As2O3 + 6Zn+ 12HNO3 → 2AsH3 + 6Zn(NO3)2 + 3 H2O. J.D. Metzger (1786) then discovered that heating of arsenic trioxide in the presence of charcoal forms a shiny black powder (arsenic mirror) over it. Carbon reduced As2O3 in this chemical reaction as 2As2O3 + 3 C → 3CO2 + 4As. V. Rose (1806) detected arsenic by applying Metzger’s test after treating the stomach samples of the deceased with potassium carbonate (K2CO3), calcium oxide (CaO), and nitric acid. S. Hahnemann (1786) mixed a sample fluid with hydrogen sulfide (H2S) in the presence of hydrochloric acid (HCl). An insoluble yellow precipitate of As2S3 formed a product of this chemical reaction [40-44]. According to the principle of this test, the arsenic-containing test sample was contacted with fresh hydrogen gas liberated by treating zinc and diluted acid. Hydrogen gas containing a small amount of arsine was then evolved. This mixture of gases was subjected to ignition, and the flame was allowed to play on a cold plate of window glass. There appeared a deposit of metallic mirror consisting of metallic arsenic. This method was also applicable for testing arsenic in inorganic materials and biological substances. This test was named the ‘Mash test’, and was extensively used in legal toxicological in- vestigations [36]. Marsh performed this chemical testing on the bio- logical samples containing arsenic and recovered arsine gas. The sample was mixed with arsenic-free Zn and H2SO4, and the presence of arsenic liberated arsine gas and hydrogen. The gas was then flowed through a tube-shaped apparatus (Marsh apparatus) and heated strongly which broke into hydrogen and arsenic vapor. When the arsenic vapor impinged on a cold surface (ceramic bowl), a silvery-black mirror-like deposit of arsenic formed on the bowl, as observed earlier in a Metzger’s test. In place of nitric acid (Scheele test), the suspect fluid in Marsh’s test was mixed with sodium hypochlorite (NaOC). The chemicalical reaction in the Marsh test takes place as As2O3 + 6Zn+ 6 H2SO4→2AsH3 + 6ZnSO4 + 3 H2O [35,44].

Marsh test was an effective and accurate way to analyze arsenic in the autopsy samples up to a level of micrograms in a concise time compared to the gravimetric method [16]. These attractive qualities made the Marsh test an immediate and broader acceptance in analytical chemistry [36]. But a significant issue with the lowest detection of a limit of the Marsh test appeared in detecting traces of arsenic naturally occurring in human bodies, then known as “normal arsenic”. It chal- lenged the correct distinction between ‘normal’ and ‘ingested’ arsenic. The concept of ‘normal arsenic’ caused a legal and scientific commotion in 1839. Another great forensic toxicologist, Orfila, strategically sup- ported the Marsh test and tried to resolve the enigma of normal arsenic bound with this test. Orfila is alleged to have differences in the solubility of normal and absorbed (poison) arsenic in the biological samples. It makes extraction of normal arsenic impossible from bones when treated with boiling water [45,46]. Edinburgh Philosophical Journal published Marsh’s innovative research findings in 1836, and later two other test articles in 1837 and 1840. Researchers from different corners of the world followed the Marsh test. Marsh’s achievement got him a medal awarded by The Society of Arts. Karl Friedrich Mohr and Justus Liebig appreciated J. Marsh efforts in developing an innovative test for arsenic detection [22]. Marsh’s successful experiment attracted chemists from every part of the world. After some time, the Marsh test also underwent a series of modifications to increase its effectiveness [36].

Marsh promoted the scientific solutions in tracing arsenic poisoning in homicidal cases. As per today’s standards, the Marsh test is an archaic one and embedded in forensic or legal toxicology history. One shouldn’t forget that this test was a turning point in tracing arsenic in the autopsy

Fig. 2. Classification of poisons according to their properties and modes of extraction [2,7,16].

R. Singh

Forensic Science International: Reports 5 (2022) 100275

5

samples and advanced forensic toxicology towards the path of devel- oping a much-needed novel procedure for extracting poisons then and now. Marsh test was the first successful step toward extraction of poisons purposely to aid the medico-legal investigations. Therefore, a true start of extracting poisons in the legal toxicology could be designated to James Marsh, who customized Scheele’s procedure to near perfection and detected arsenic. Orfila after a few years, thoroughly exercised the Marsh test in extracting arsenic from human organs.

1.4.4. Mathieu Joseph Bonaventure Orfila (1787–1853) M.J.B. Orfila was born on 24th of April 1787 in Mahon (Minorca),

Spain. In the nineteenth century, he was a well-known medical analyt- ical chemist, and one of the founder fathers of legal toxicology. His scientific work formed a rational link between toxicology science and the judiciary. He also classified the poisons in a medico-legal context. His experimental findings were a milestone in the progress of forensic toxicology. He took an interest in poisons, particularly arsenic, and started performing experimental toxicology using animals. His expertise and opinion were sound enough to help the court of law solve medico- legal cases. One of Orfila’s testimonies was considered a matter of debate in French medico-legal society [47–49]. According to the liter- ature [50], Prof. Dr. Muaoz, in a Spanish-language biographical tribute to M.J.B. Orfila, published in 1956 in Quito, Ecuador, called him the "founder of modern toxicology". A biographical sketch revealing Orfila’s excellent career appeared at his death [51,52]. He remained in France until his death on the 12th of March 1853 [49]. His outstanding work on legal medicine ‘Traité des poisons, tirés des reégnes minéral végétal et animal; outoxicologie générale, considérée sous les rapports de la physiologie, de la pathologie et de la médecinelégale’ (Spanish) was published in two volumes (1814–1815) in Paris which was then trans- lated in English [48].

1.4.5. Analysis of arsenic poisoning Orfila was the first toxicologist to systematically use chemical anal-

ysis on autopsy samples as legal proof of poisoning. In 1839, Orfila also effectively exercised the Marsh test (1836) to identify arsenic extracted from human tissues [2,47]. Applying his scientific skill, Orfila used to work on the clinical data of suicidal poisoning with arsenic, especially in the case of Soufflard (1839). Orfila then described his experiences and discussed the problem of arsenic absorption [Mateu Orfila, De l’em- poisonnement par l’acide arsénieux, Bulletin de l’Académie Royale de Médecine(1839) & L’’Expe’́rience (1839) [46].

Orfila’s amazing expert testimonies in legal trials:-The courts largely witnessed several novel scientific strategies aiding forensic medicine in solving poisoning cases during the nineteenth century. Orfila’s scientific inputs are highly spoken in Nicolas Mercier (1838) and Lafarge affairs (1840) cases during the thumping period of arsenic poisoning. Orfila started his investigation on the provided exhibits of the deceased applying a modified Marsh apparatus and found arsenic-like black spots. Scientific opinion established the death of Nicolas Mercier with some arsenical preparation but, few genuine queries gave birth to a contro- versy against the scientific technicalities of used tests [46].

Orfila’s role as a scientific expert in the Lafarge affair is a matter of discussion from the forensic point of view. In 1840, Charles Lafarge’s mother accused her daughter-in-law Marie Lafarge of poisoning her husband with arsenic. It was supposedly a case of family conspiracy over wealth. Orfila submitted the expert’s technical ignorance in conducting the tests. Because of the contrary results, the court called Orfila to settle down the issue with a scientific means. Orfila declared the presence of arsenic in the exhibit samples of the body to the exclusion of all control samples [22,46,53]. Many believed that Marie was a pray of injustice and weak scientific evidence. Orfila’s experiments involving the Marsh test also received uncertainty and criticism from several toxicologists, particularly the British, for his explanatory experiments differentiating ‘normal arsenic’ from deliberate ones [46]. Orfila didn’t participate as an expert witness after 1843.

It was a significant phase of his medico-legal expertise but in isola- tion. Orfila made a considerable contribution to extracting poisons from the biological samples for legal investigation of poisoning cases. He used conventional testing but innovatively to the best of his scientific skills. His name in the field of forensic toxicology deserves colossal apprecia- tion. Orfila was a medico-legal celebrity of his time. He still lives in the recent articles covering prehistoric legal toxicology.

1.4.6. Jean Servais Stas (1813–1891) Jean Servais Stas was a Belgium analytic chemist who invented the

basic ‘Stas’ procedure. He was born in Louvain, Belgium, on the 21st of August 1813. He served medico-legal toxicology with an outstanding investigative work on the isolation of nicotine alkaloids for the first time in 1850. It was a qualitative and quantitative analysis. Stas’ extraction method is a prototype and saw few modifications, especially the im- mediate and defining one made by Julius Otto (1856), which further advanced this method to extract several non-volatile acidic and alkaline compounds. Stas’s experiment was hereafter known under the name the Stas-Otto method. It is a customary extraction process used in forensic and other science analytical laboratories. Color/spot test and crystallo- graphic properties further identify separated alkaloids [2,54–60].

1.4.7. Aalkaloidal poisons in medico-legal toxicology According to a recent article, ‘Poisoning crimes and forensic toxi-

cology since the eighteenth century’, the isolation and identification of alkaloidal in particular was a critical and challenging issue observed by the analogous chemists practicing ubiquitously to solve mysteries of criminal poisoning during the nineteenth and early twentieth century [6]. This article also quoted comments given by a French physician and criminologist A. Lacassagne (1843–1924), who studied trends of crim- inal poisoning in France for the period 1825–1900 and generated a le- gally significant database. Lacassagne appreciated several convectional toxicological experts, including Orfila M.J.B. and Stas J.S. for their remarkable legal expert opinions contemplating ‘no poison detected means no poisoning’, which is unlikable in the modern scientific anal- ysis [61]. During the mid-nineteenth century, a Belgium chemist Stas J. S. first time developed a method for extracting nicotine from biological samples using a solution of acetic acid in warm ethanol. Later in 1856, a German chemist Otto F.J. modified this procedure and quickly applied it to isolate other alkaloid poisons e.g. colchicine, coniine, morphine, narcotine, and strychnine.

1.4.8. Recognition of Stas extraction method in the lights of Bocarḿe case While serving at Brussels, his participation in the capacity of an

analytical expert in the Bocarḿe case (1850) is an outstanding chapter in the history of forensic toxicology. He played a leading expert role in this murder trial, leaving a lasting influence on legal toxicology. This case revolved around Belgian Count Hypolyte Visart de Bocarḿe (1818–1851), who was legally charged for murdering his brother-in-law Gustave Fougnies by poisoning him with nicotine (then undetectable) containing poisonous potion in a castle at Bury (Belgium) in November 1850. Bocarḿe‘s wife was also involved as an accused in the case [62]. Bocarḿe knew about preparing the nicotine alkaloid and its immediate action on the human body [63]. Preliminary autopsy findings suspected his death of consumption of any corrosive liquid acid. Autopsied sam- ples were preserved in ethyl alcohol for chemical analysis. A further legal investigation was pointing to the use of tobacco leaving isolated nicotine to poison Fougnies, but concrete scientific evidence was lacking to prove the same.

Stas was then working as a Professor of Chemistry at Brussels. He was called by the examining judge Bemelmans to conduct a chemical anal- ysis of autopsied samples (stomach, intestines, lung, and a few others along with the preservative) of the deceased. Stas’s preliminary scien- tific inputs indicated using some plant alkaloidal poison and ruled out corrosive acid [59]. Applying his masterly skills, Stas performed chemical analysis on the case exhibits by adopting his novel method of

R. Singh

Forensic Science International: Reports 5 (2022) 100275

6

detecting nicotine [56]. Experimentation on extracts using sulfuric acid and nitric acid indicated the presence of a nicotine-like volatile alkaloid in samples. While testifying in the court, Stas claimed to have isolated a substantial amount of nicotine in the autopsy samples. The results were convincing enough for all the jurists, including the judge to declare death by tobacco-like substance. There was a lack of published work on nicotine extraction at that time; therefore, Stas had claimed the un- availability of previous research reports on this kind of poisoning [56, 64].

In this way, Stas had the first time isolated nicotine like alkaloid poison using a solution of acetic acid in warm ethyl alcohol. Stas also enlightened that ethyl alcohol used as a preservative of viscera played a significant role in keeping nicotine trapped in the autopsy samples under acidic conditions. Alcohol not only deproteinized the sample but also acted as a ‘keeper’ to preventing nicotine volatilization. Stas deprotei- nized the biological specimens with ethanol following solvent extraction with diethyl ether [2]. This case took an interesting twist when Orfila challenged Stas’ findings, claiming practical unfeasibility of the used method because he had already refuted such isolation [63]. An official debate in the press and the court gave Stas scientific and legal credit for his novel method of extraction [62]. The court satisfactorily and scien- tifically adjudged Bocarḿe guilty of murder. Stas published his influ- ential results in 1852 that left a significant impact on analytical post-mortem toxicology [59].

The basic Stas extraction procedure has been followed from the ‘Manual of the Detection of Poisons by Medico-Chemical analysis’ by Otto, Fr. Jul., translated from the German, with notes and additions by William Elderhorst, New York: Bailliere, 1857 [65]. This procedure in detail has been explained by Otto FJ (1856) [66]. Many other workers made necessary improvements in the primary Stas-Otto procedure. Stas was a designated toxicology expert in several legal court trials, and he kept contributing with other scientific deeds. His academic achieve- ments confirm his brilliance in the scientific domain. Stas died in Brussels on 13th of December 1891 [56]. Recent forensic toxicology books might not have any deserving applause for the detailed contri- bution of Stas as a chemico-legal expert in legal toxicology. However, right from the nineteenth century, the fundamental of isolating the alkaloidal poisons developed by Stas for aiding the medico-legal toxi- cology has effectively paid a major part in breaking boundaries of analysis in post-mortem or forensic toxicology.

1.4.9. Friedrich Julius Otto (1809–1870) F.J. Otto, a founder of the ‘Stas-Otto’ extraction method, was born on

the 8th of January 1809 in Grossenhain, Germany. He was an analytical chemist, pharmacist, technologist, and public health official. Apart from the pharmacy, he also researched food chemistry, technical chemistry, and toxicological analysis issues. In 1829, Otto’s interest in pharma- ceutical chemistry brought him to the University of Jena. He then worked on analytical chemical technology and got a doctorate at Jena in 1832 in acetic acid manufacture and testing. He satisfactorily contrib- uted to the scientific field with his impressive published experimental work on agriculture and pharmacy [67]. He died on the 12th of January 1870 in Brunswick, Germany.

1.4.10. Development of Stas-Otto procedure and notable modifications It is now understood that the basic ‘Stas’ (1851) and the ‘Stas-Otto’

(1856) are two different methods. The ‘Stas-Otto’ (1857) was the first version of modifying the ‘Stas’ procedure while other changes made in the basic ‘Stas-Otto’ procedure, are known as modified Stas-Otto methods. The basic procedure, the parental form remains almost the same as in ‘Stas-Otto’ (1856). Slight changes were recorded at quite a few steps of this long procedure. Otto worked on improving the earlier developed procedure by Stas (1851) to isolate the alkaloids from bio- logical samples. Otto made the foremost modification in the Stas extraction procedure in 1856. According to an extract taken from ‘Manual of the Detection of Poisons by Medico-Chemical analysis by F.J.

Otto, translated from the German, with notes and additions by Eld- erhorst in 1857 the Stas method was slightly modified and stated as “The substances are treated with alcohol and tartaric, or oxalic acid; the extract is concentrated by evaporation; the resulting aqueous solution is separated by filtration from the insoluble matters; the filtrate is mixed with a solution of caustic soda, and distilled. The alkaloid will be found in the distillate. For its separation, two ways may be followed. The distillate is either agitated with ether, and the ethereal solution exposed to evaporation; or it is neutralized with oxalic acid, the liquid concen- trated by evaporation, and the residue treated with caustic soda and ether when also an ethereal solution of the alkaloid is obtained” [65].

In the abstract, Otto treated the aqueous extract of the tissue with ether to remove other non-alkaloid substances in the ether layer. It resulted in added extraction and detection of neutral and acidic poisons. The procedure begins as usual by treating the sample with ethyl alcohol and tartaric/oxalic acid, and the extract is concentrated with evapora- tion. The addition of a highly constructive step improved the versatility of the Stas procedure and renamed it the Stas-Otto procedure generally applied in Systematic Toxicological Analysis (STA), especially in “gen- eral unknown cases” to detect and identify toxic compounds. The con- ventional toxicological analysis in forensic medicine contained few accurate methods for detecting unlike poisons; however, mostly appli- cable when prior circumstantial information or certainty about poison was available. There was a lack of a systematic method for the detection of any poison. Professor Otto made an excellent attempt to overcome this toxicological hurdle by developing a general analytical procedure to ascertain the nature or otherwise the division of the substance under examination without any prior hints. Otto published his findings in 1856 [64], and soon his research work was translated by Elderhorst [65]. This scientific achievement recognized F.J. Otto a distinguished analytical chemist in the discipline of legal toxicology [58,65,67].

The timeline reveals several valuable modifications in the funda- mental Stas-Otto process made in the past, especially by Autenrieth, Dragendorff, Bamford, Magnin, and Zappi [67]. These modifications were madeto bring the ease to the utility of this process. It’s difficult to explain all such modifications in detail; however, according to my observation, medication presented by Bamford (1947) is imperative to discuss. This modified method was also cited in another analytical utility book ‘Handbook of analytical chemistry’, First edition, Section 13, Methods for the analysis of Technical Materials, Toxicological analysis by B.P. Parker and P.L. Kirk edited by Meites [68].

With added revisions in the Stas-Otto method, Bamford (1947) [16] involved isolation of (i) non-basic organic poisons (poison classification group (v) soluble in alcohol and a little in water or dilute alkalis with the help of acidified solutions shook with ether, chloroform, or other immiscible solvents, and (ii) basic organic poisons (poison classification Group 6) with immiscible solvents from aqueous solution in the presence of alkali. Consensually, Bamford (1947) observed the Stas-Otto process, a long (usually 4–5 days) tedious mode, but all the steps were considered not customarily essential in exceptional cases. Bamford (1947) also devised a scheme for extracting non-basic poisons (organic poisons other than alkaloids) from the residue obtained by evaporating alcohol in the Stas-Otto process. Hot water-dipped residue weakly acidified with H2SO4 was extracted sequentially with (i) light petroleum ether extract (for many oils), (ii) ether extract (hypnotics or soporifics), (iii) chloro- form extract (glycosides), and (iv) some specific solvent such as ethyl acetate or amyl alcohol.

1.4.11. Alan Stewart Curry (1925–2007) A.S. Curry was a highly influential English forensic toxicologist of the

twentieth century. He was born on the 11th of October 1925 in Black- pool, UK. He earned a doctorate from Cambridge and then started serving as a forensic chemist at the Home Office London. His acme performance in forensic toxicology is immensely remarkable which earned him a reputation as an internationally renowned forensic expert. Curry was a brilliant forensic toxicologist with several noteworthy

R. Singh

Forensic Science International: Reports 5 (2022) 100275

7

publications to his credit. He published research work on emergency hospital toxicology and post-mortem toxicology to develop rapid pre- sumptive diagnosis and treatment of poisoning. Serving at his best, Dr. Curry died on the 20th of August 2007 in Berkshire, England [69].

1.4.12. Curry’s inputs on extraction methods From the mid-twenty century up to now the medico-legal community

has greatly benefitted from value contribution comprising reviews and experimental work published by Curry. He keenly observed the earlier work related to isolation and identification of drugs and poisons from autopsy samples. Curry exhibited his skill of presenting the research through his articles published in highly reputed journals i.e. Nature (1954), and Journal of Pharmacy and Pharmacology (1955, 1959 & 1960), etc. His research theme was based on the isolation and detection of the alkaloid extracts in forensic toxicology. His review article on toxicological analysis published in 1960 is worth reading in forensic toxicology. Curry reviewed several critical systematic chemical ap- proaches (including his own) in forensic toxicology analysis. He dis- cussed three methods of isolating organic solvent-soluble poisons from viscera and biological fluids, including urine. He addressed the utility of organic solvent for extracting poison and drugs from small quantities of biological materials in buffered aqueous solutions [70]. Curry and Phang (1960) then devised an efficient ethanol continuous extractor that worked under the reduced pressure to operate the Stas-Otto process effectively, especially for the labile alkaloids in a reduced time of 4–5 h. The study helped solve the issues associated with a large amount of ethanol used as a preservative in cases of organ putrefaction at high-temperature conditions [71]. Another contribution made by Curry and his associates helped in the rapid emergency hospital toxicology by applying simple preliminary tests on urine, blood, and stomach aspi- rates. The solvent extracts were then subjected to Thin Layer Chroma- tography (TLC) and Ultraviolet (UV) spectrophotometry for further identification [72].

Curry’s highly circulated and cited book ‘Poison Detection in Human Organs’ published in three editions in 1963 [73], 1969 [74], & 1976 [75], seems a highly considerable chronicle of extraction methods in forensic toxicology. Described below are the routine methods of poisons extractions in post-mortem samples, including liver and alimentary ca- nals by using ‘Sodium tungstate precipitation’ (R.H. Fox) [76] and ‘Aluminum chloride’ method as an alternative method [77] as the first method of treating liver sample for toxicological analysis. The ether and aqueous layers in these methods are treated to obtain i) strong acid fraction for salicylic acids, ii) weak acid fraction for barbiturates and PCM, etc iii) neutral fraction for carbamates, iv) alkaloids & base fraction for nicotine, etc, and v) morphine fraction where the extract was redis- solved in ethanol and extracted with chloroform: isopropanol (4:1) [75].

Literature confirms that Dr. Curry was an outstanding pillar of forensic toxicology in the recent past. A deserving scientific brilliance in forensic toxicology admired Dr. Curry with honor on several occasions. Playing a vital role in founding the International Association of Forensic Toxicologists (TIAFT), and acting as its first secretary and then the president is great achievements in the credit of Dr. Curry, furthermore, TIAFT in revert saluted his legacy in forensic toxicology and declared a highly prestigious ‘The Alan Curry Award’ for the members with emi- nency. He was awarded with a Fellow of the Royal Society of Chemistry, the Royal Society of Pathologists, the Belgian Pharmaceutical Society, and the Indian Academy of Forensic Sciences [69]. A timeline of pio- neers included in this discussion and their notable contribution to legal toxicology has been shown in Fig. 3.

2. Discussion

Since ancient times, murder by poisoning has been considered a dreadful act mainly driven by bad intentions. The medico-legal science dealing with toxicological analysis is old enough and comes from enormous ancient times. An autopsy is a considerable tool in the medico- legal practice. A review of authentic records of medico-legal toxicology produced noteworthy literature on criminal poisoning and extraction methods from countries like Spain, Belgium, Germany, and England. Several pivotal judicial proceedings during the eighteenth century were a landmark in edifying collective efforts of two professional branches i.e. forensic medicine and forensic toxicology in solving crime poisoning cases. The present article also glanced at scientific stories associated with the development of forensic toxicology from the beginning of the nineteenth century up to the end of the twentieth century. According to Saferstein (2007) secret of poisons in different autopsy samples can be uncovered by applying systemic or specific extraction method proced- ures demonstrated in a laboratory analysis worksheet. Application of a profound knowledge of poisons and accurate extraction method can easily help solve complex toxicological cases (by successful isolation), but in failure of doing so the forensic outcome could be misleading and changeling in the legal proceedings [4].

Occasionally, the much desired but undecipherable factual scientific information can be of immense utility for the concerned at any analysis and research development. The medico-legal and forensic domains al- ways need elaboration on the origin, importance, technicalities, impli- cations, and current status of such procedures for their legal demonstrations. There are several published items defining applications of the extraction procedures in the scientific fields, but forensic science applies these techniques in casework, thus somewhere lacks substantial participation in the literature. Given the proper explanation of several forensic implications related to the classification of poisons and their

Fig. 3. Timeline of Pioneers and their notable contribution in legal toxicology.

R. Singh

Forensic Science International: Reports 5 (2022) 100275

8

extraction, the discussion includes fowling points of review:-

2.1. Satisfactory classification of poisons in the current scenario

Classification of poisons has numerous parametric versions but usually remains limited in forensic toxicology. Depending on the in- terests and needs of the classifier, the toxic agents could be classified according to different parameters e.g. use, source, effects, physical state, poisoning potential, etc. The single mode of classification might not be appropriate to the wholesome spectrum of toxic agents; therefore, the amalgamation of classification systems or depending on other factors may help to illustrate the best suitable system for a defined purpose. In general, the chemical and biological properties of the toxic agent, along with their exposure characteristics could be considerable and helpful in classifying poisons for legislative and toxicology purposes. Large poisons containing groups cab be divided into sub-groups to apply more convenient and specific extraction methods.

Literature of forensic toxicology also confirms the use of several methods (dry ashing, wet digestion, enzymatic hydrolysis) for extracting metallic poisons, dialysis for toxic anions, and distillation for volatile poisons. It lays a scientific basis for classifying the method for separating a toxic substance from the matrix/biological specimen (body fluid or a solid tissue) in which it is embedded [2]. While approaching the initial stages of any toxicological analysis, an expert is generally concerned with removing the crude and other products of toxicants from biological samples. Therefore, it becomes convenient to apply a broad chemical classification covering volatiles, metallic poisons, and non-volatile organic poisons based on this extraction process. The systems of poi- son classification have recorded a few essential and timely updates. We have relative standard references to follow in answering the scientific and legal queries. The concern of this article is also with the advanced poisons, especially radioactive substances e.g. polonium-210 [78]. Bio- terrorism has been a burning topic for the last few decades. Stll, the availability of cases remains far away from the society and limited to quite a few areas to the rarest occasions on. Therefore, it is felt to slightly upgrade the current classification systems of poisons from every important aspect of the legal sciences.

2.2. Expert understanding of adopting ‘general’ and ‘specific’ extraction procedure

As reported that no single extraction could be an exaggerated one for the best possible recovery of all types of toxicant substances [79]. Sci- entific reports depict specific as well as a general/broad approach for the extraction of poisons from the autopsy sample. Marsh test was a specific one for detecting arsenic poison in the biological sample, whereas Stas-Otto was dedicated to the separation and purification of several organic alkaloids. Records of analytic toxicology have proved that pre- ceding forensic toxicologists knew that unknown suspected poison should undergo a systematic way to confirm the identity of the general toxic substances. Therefore, the classification of poisons given in ‘Casarett and Doull’s Toxicology: The Basic Science of Poisons’ was explanatory to the methods of separating the toxic agents accrued in the biological specimen. The selection of an accurate extraction method is a crucial point of analysis; otherwise, separating actual analytes in suffi- cient quantity could be difficult for the dealing expert. Methods for separation of toxicants from biological samples have long been chal- lenging to analytic toxicologists therefore, an effective scientific strategy is a crucial matter of expertise [2,4].

Blyth and Blyth (1906) commended and recommended the original process of Stas and its beneficial modifications in identifying alkaloid/s alongside their capabilities of disposing of interferences such as fats and salts, etc., and efficiency of dissolving as little as possible of foreign substances. He also added that the Stas-Otto process, Kippenberger process, and Dragendorff are well-known general processes for sepa- rating alkaloidal substances from the organic matters, but somewhat a

combination of these three processes is more fruitful than using indi- vidually for an effective operation [63]. Analytes are generally found in a solution of protein and other cellular-bound constituents. These situ- ations can pose implications in their pure separation quantification. A thorough study of isolated metabolites was considered in identifying the parent toxicants [2,80].

Generally, the non-volatile poisons form a larger group where vogue extraction can be applied, including using suitable solvents (e.g. Stas- Otto) and precipitation or other methods of removal of protein (e.g. Ammonium sulfate), leaving the toxic substance in solution [81]. Removal of verities of organic drugs from association with viscera is accomplished by applying the Stas-Otto method which is accepted as a universal method of choice for isolating an “unknown” poison [82]. An extract is taken from ‘Handbook of Analytical, which proposed a ‘sys- tematic approach to isolate the toxicological substances the first pref- erence by the analyst, especially when the analyst is trying to isolate an unknown substance in micro quantities in the given sample. Two sys- tematic schemes for extracting poisons from viscera include steam distillation (acidic & alkaline), and solvent extraction (with immiscible solvents) forming aqueous and solvent (ether and chloroform) fractions [68]. According to Curry (1976) extraction approach doesn’t carry any universal but rule rather negative test results from one extraction method following other methods in sequence. An incomplete under- standing of whole analytical methods and procedures could certainly impede the results in poison detection [75]. The author of a critical review on extraction methods reported that selecting a suitable method of extraction depends on the exact type of drug/s a toxicologist is tar- geting to isolate [83]. He also referred to supporting work published by several other authors [17,74,75,84].

A comprehensive forensic toxicological analysis undertakes five steps i) selection, ii) extraction; iii) separation; iv) identification, and v) quantization of drugs/poisons and their metabolites; however an expert must understand that any step/s might be optionally passed over depending upon analytical requirements e.g. extraction as in case of direct immunological testing [5]. According to Saferstein (2007), a planned and properly devised extraction procedure can make out suc- cessful isolation and detection of toxicants along with their metabolites from the matrix (fluids or tissue). An accurately applied extraction method can help to solve complex toxicological cases but in the failure to do so, the forensic outcome could be changeling in legal proceedings [4].

2.3. Exaggeration of auxiliary analytical knowledge

Tully revealing a catastrophic status of forensic science pointed to- wards a severe shortage of expertise, toxicology in particular in England and Wales [85]. In another way, it is evident that the toxicological analysis is not simple as it seems. The chemistry of poisons and their fate inside the body is a broader area to understand. Still, the toughest is to detect them in a minute quantity of milligram/nanogram level from the autopsy samples. Occasionally the concentration of toxic substances might fall or disappear in the biological samples as the time progresses after the autopsy for several reasons. Many may be unknown to the expert as well. Furthermore, the biotransformation can also transform one form of a toxicant into another one depending on pharmacological factors. An effective extraction strategy could be an effective approach for better toxicological interpretations. According to Finkle (1982), drugs in general also play poisons in toxicology cases. Therefore, the expert knowledge of extraction, identification and quantification of drugs and metabolites in the biological samples to near perfection is an evident qualification to understanding toxicology as well as settlinge legal issues [5].

Forensic toxicology encompasses several scientific impediments but is fixable at an expert level by being cautious and thorough, both practical and theoretical. Right from the beginning, toxicological research is based on crucial data generation using analytical chemistry,

R. Singh

Forensic Science International: Reports 5 (2022) 100275

9

and interpretation using this data within a particular case context. Therefore, it would be a little difficult for the forensic toxicologists to brush their scientific knowledge and skill unless being conversant with momentous editions of e.g. ‘The detection of poisons and powerful drugs [86], ‘Poison Detection in Human Organs’ [73–75], Clarke’s Analytical Forensic Toxicology [7,17] and Toxicology, the Basic Science of Poisons [9,18,19], and other essential literature reports helpful in understanding toxicology analytical strategies. According to the author’s understand- ing, there are various general and specific or technical points of concern for performing effectively extracting toxic substances from the forensic samples.

A) General points: - These points though directly or indirectly may be under the consideration of experts performing extraction of toxic substances from the autopsy or other biological samples: -

i) Analysts must know that an autopsy, in general, is a one-time opportunity to collect as many specimens as needed to com- plete the toxicological analysis; therefore, they must be cautious enough to use the appropriate quantity of provided biological samples. A certain amount of substance might be lost while going through tedious extraction steps.

ii) A thorough appraisal of all the submitted documents e.g. post- mortem report, first information report, and any other relevant information can direct towards using a specific approach in extracting toxic substances. For example, a victim before death could have been hospitalized and might have been given anti- dotes or other curative drugs; therefore, a careful assessment of such substances must be considered while adopting the extraction method. Concepts and chemistry of antidotes e.g. anti-snake venom in snakebite, atropine sulfate in pesticide poisoning death, and many others, should be clear while interpreting the results.

iii) It should also be kept in mind that the poisons found in the stomach and that extracted from other organs can lack correla- tion on several occasions due to disintegration and absorbance of poison in blood circulation once leaving the stomach.

iv) The concepts of post-mortem re-distribution (drug movement and concentrations variation within the body after death but before autopsy), properties, mechanism of action, biotransformation, and classification of toxic substances should be in the expert domain knowledge because these points help in an effective extraction. It also aids in choosing the correct biological samples for a particular class of drugs/poisons, especially when an available biological sample is limited, putrefied, from the burnt and electrocuted bodies, as such circumstances could surely diminish the efficacy of extraction and detection of toxic agents, if any.

v) Applying extraction procedures on putrefied samples can also be a matter of concern. These samples could have unlike material depending on their changing biochemistry with the passage of time and deterioration of bio-materials. For example production of ethyl alcohol in a dead body through the topic is a chronic point of discussion. Scientific reports disclose that ethyl alcohol might circumstantially be produced after death by microbial ac- tivity and glucose fermentation, especially after the corpse’s decomposition [87,88].

vi) Preservation of biological samples, especially viscera, is an important aspect of forensic toxicology investigation, especially more essential in a tropical country where mercury shoots up to extreme during the summer season and may hasten putrefaction of biological material. Putrefaction of viscera can exert a destructive effect on contained poison (e.g. Malathion) by split- ting them into simpler non-toxic degradation products or losing them forever (e.g. Atropine). Using preservatives is important for the long-term protection of autopsy samples. Expert knowledge

about the use (if any), nature, and quantity of preservatives is a crucial aspect of the extraction procedure. Consequently, the requiremt of preservative sample and other biological exhibits for toxicological analysis is mandatory. Rectified spirit is a consid- erable preservative now and always except when a case of poisoning by alcohol, carbolic acid, Paraldehxde, etc. Henceforth, viscera suspected of alcohol poisoning is usually preserved in normal saline (0.9% w/v sodium chloride) solution, while use rectified spirit can be made in case of non-alcohol poisoning. The brain is also fixed in 10% formalin for one week or so. There are verities of preventives for bio-fluids e.g. use of antioxidants (So- dium fluoride, Sodium nitrite, etc.), and anticoagulants (EDTA and potassium oxalate, etc). Preservation of urine can interfere with the concentration of Phenobarbital substances. Apart from using a wide range of preservatives, refrigeration is another best way of preserving a few biological samples.

vii) The concept of volatile drug evaporation and “salting-out” effects should be in mind. The usese of disposable Pyrex glass tubes, especially for blood, and other glass containers can help to sort such problems. Lungs in case of volatile poisons are preserved in a nylon bag, and polythene bags shouldn’t be used for this purpose as they can leak volatile poisons.

viii) Heat and fume producing reactions happen in processing extraction procedures e.g. steam distillation. So be aware of the toxic hazards (if any) associated with such chemical reactions. Liberation of phosphine gas from viscera processed for extraction in case of aluminum/zinc phosphide poisoning could be life- threatening. Use of safety measures e.g. eye spectacles, hand gloves, face mask, apron, and working under fuming hoods are essentials for conducting toxicological analysis. Defined bio- safety levels must be adopted for personal and social wellbeing.

ix) Expert knowledge on poisons and associated new extraction strategies should be kept updated through advanced as well as convectional scientific periodicals to remove occasional impedi- ments in the extraction scheme.

A) Specific points: - Likewise, many specific or excessively technical points help in a better extraction strategy.

i) Experts should understanding systematic toxicological analysis (STA), especially in detecting unknown/unsuspected toxic com- pounds. Always be fully conversant with the principles of sample preparation, especially for the alternative samples, and the lim- itations of extraction methods.

ii) All experimental conditions, including room temperature, heat- ing conditions, storing conditions, the sample quantity to be used, and pre-treatment (maceration and homogenization) should be well-known to the expert.

iii) Experts must understand the basics of ‘back extraction’ and pH extent in the toxicological extraction practice. Many toxic sub- stances, including drugs in particular, can be ionized in a specific pH range. This property of drugs helps their back-extraction which is an vital attribute of liquid-liquid or solvent extraction. The basic extraction process begins by adjusting the pH (by salting) of the aqueous phase so that the analyte turns neutral in form. Consequently, the analyte migrates into the organic portion and gets dissolved therein. Generally, the organic acids and bases can be less soluble in water than salts. The Back-extraction step involves mixing the organic phase with the aqueous phase at pH appropriate for ionizing the analyte, which is duly soluble in the aqueous phase, and possible to be back-extracted. This procedure is suitable for the simple and effective separation of analyte from non-polar and several polar impurities [89]. Thus back-extraction improves the selectivity of the extraction procedure at varying acidic and alkaline conditions. Therefore, the basic substances would extract into the organic solvent at high pH, and vice versa.

R. Singh

Forensic Science International: Reports 5 (2022) 100275

10

The non-polar drug would extract back from the organic solvent into an acidic medium, and vice versa for the acidic drugs. Though there is no limit to the attempts of back extraction steps, absolute analyte concentration keeps decreasing with every go as per re- covery achieved every time.

iv) The presence of fats and proteins in biological samples to be executed for extraction can pose a significant problem in analytical toxicology [90]. Fat is a crucial hindrance in the extraction procedure; hence, removing fat should be the first step in the extraction procedure as described in ‘Handbook of Analytical Chemistry’ [68]. At first, the macerated tissue is mixed with 5% acetic acid saturated with ammonium sulfate, and heated in a boiling water bath for 30 min. Then the solution is filtered on a paper pulp pad in a sintered-glass Buckner funnel using such suction that fat is not pulled through. Washed twice with hot 5% acetic acid aliquots and combined with the filtrate and washing bracket [91]. In the second method, the macerated sample is shaken with a suitable solvent and a large quantity of 95% ethanol for one hour in a sealed container. Adding anhy- drous sodium sulfate (100–200 g) prevents the formation of two phases. Then separated the liquid from the solid, and removed ethanol by washing it with water. Evaporated the organic phase to 2 ml, added 0.5 g Celite, and again evaporated to dryness. Dissolved the residue in acetonitrile and prepared a solution containing 40% v/v acetonitrile and 60% water. Passed the so- lution in last through a polyethylene alumina column to remove fatty material, leaving the effluent for analysis [92,93].

v) Properties and selection of extracting solvent/s are crucial for solvent extraction so are extraction temperature and duration for effective solvent extraction. Rule of ‘like dissolves like’ when solvents having a polarity near the solutes’s polarity are likely to perform better, and vice versa. Ethyl alcohol and methyl alcohol with plentiful assay have been considered the universal solvents in solvent extraction for different analytes, especially for phyto- chemicals [94]. Using solvents like chloroform and petroleum ether is highly effective for alkaloidal extraction as referred Stas-Otto procedure. According to Tompsett (1968), diethyl ether and ethyl acetate are good general solvents, whereas benzene and heptane are selective solvents, especially in the case of urine. Diethyl ether though readily evaporates due to its low boiling point but it is a highly inflammable one. Ethyl acetate is not particularly selective in its action because of its insolubility in other organic solvents. Benzene can present an intense and interfering ultraviolet spectrum, while heptane is particularly useful in spectrofluorimetry of drugs like phenothiazine. He also added that chloroform doesn’t have a good effect as extracting solvent for Morphine in comparison to neutral aqueous/aqueous solutions containing ammonia [90].

vi) It is equally important for the expert to be conversant about the referred inferences including, odor, color, crystal formation, etc while the extraction is in progress.

2.4. Coherent of conventional extraction

2.4.1. Methods & future perspectives The earliest toxicologist experts were the analytical chemists who

developed analytical techniques by which they could isolate and iden- tify offending poisons from post-mortem samples. The trend of chemist turning toxicologists by applying their analytical skills to investigate poisoning cases continued up to 20th century. Ordinary as well as modified versions of the Stas-Otto process have proven their worth in many famous cases in the arena of medico-legal studies. It is still a

standard method of extracting organic poisons in forensic and other scientific fields. The Stas-Otto method has been endorsed for more than one and a half-century and is still effective. Off-course, modifications made this procedure better to cover wider extractable substances. Several methods used today to extract organic material from biological and other samples have fundamental similarities with the parent Stas- Otto method. Therefore, from my point of view, it is almost impos- sible to complete a worthwhile tome on forensic toxicology without recitation the Stas-Otto procedure primarily covering chemical extrac- tion and analysis of autopsy samples in poisoning cases. From a technical point of view, the Stas-Otto procedure has also faced quite a few positive criticisms, especially regarding its limited practicability, tediousness, and lengthiness [82,95–97]. Similarly, protein precipitation methods e. g. Ammonium sulphate[98] and sodium tungstate [75,99] felicitated forensic toxicological extractions but lacked suitability at a few tech- nical points [96]. The significant contribution of the ‘Stas-Otto’ pro- cedure sometimes seems to have been little omitted and forgotten long back since the emergence of chromatography aid in forensic toxicology. The methods usually employed to isolate organic poisons are mainly based upon the standard Stas-Otto process. Tissue samples have been considered the most complex matrices to extract toxicants (drugs and pesticides), particularly for their accurate quantitative analysis [100, 101]. Extraction methods, in general, include distillation and solvent extraction methods. Maceration, percolation, and reflux extraction are some of the conventional extraction methods that usually include organic solvents and long extraction time. Literature also reports the use of Manske’s process, Kippenberger’s process, soxhlet extraction, pres- surized solvent extraction, negative pressure cavitation extraction, pulse electric field extraction, etc as other types of systematic, particularly for the extraction of drugs and their metabolites from biological materials [102]. Pressurized liquid extraction (PLE), supercritical fluid extraction (SFC), andmicrowave-assisted extraction (MAE) are the modern or greener extraction methods generally applied in natural products extraction. They have the advantage of shorter extraction time and higher selectivity [94]. The analysis of tissues by TLC was also consid- ered to be more difficult due to the extraction and chromatographic problems related to a different complex matrix. Classical procedures utilizing protein precipitation, acid hydrolysis [103], enzymic digestion [104,105], coupled with specialized extraction techniques [106] and other complex development systems103 were suggested to remove tissue interferences, enhance recovery, and improve chromatographic sepa- ration. But time consumption and the requirement of large amounts of samples are the main disadvantages of using these methods [107].

The sample preparation step is crucial for accurate and sensitive forensic toxicological analysis, mainly when relevant analytes in the biological samples are in complex and trace forms. The current world- wide scenario of analytical forensic toxicology seems deeply involved in ‘microextraction techniques for extracting substances of forensic interest from biological samples. The volume of the extracting phase in the microextraction technique remains smaller in comparison to the volume of the sample, and the extraction of analytes usually remains non- exhaustive [108]. With the progression of time, various versions of microextraction-based sample preparation techniques in green chemis- try have been swiftly evolving to knockout the use of solvent in large volumes and better analysis [109]. In recent years, the use of micro- extraction techniques e.g. solid-phase microextraction (SPME), micro- extraction by packed sorbent (MEPS), and liquid-based microextraction such as single drop/hollow fiber-based liquid-phase microextraction and dispersive liquid-liquid microextraction (DLLME) have been offering their promised utility in forensic toxicology analysis for developing faster and more ecological analysis [110–113]. Simplicity and low cost also favor wide acceptance as well as practical availability of such

R. Singh

Forensic Science International: Reports 5 (2022) 100275

11

techniques in analytical laboratories [114]. It is pertinent to know that the use of conventional lengthy liquid-liquid and protein precipitation-based extraction procedures in forensic toxicology are still contained in the formal worksheets of forensic science laboratories. They are an integral part of the toxicological analysis, especially when an approach is applicable for widespread or unknown analytes. It is acceptable that the new phase of microextraction is also workable and useful, but the eradication of conventional methods of extraction is not easy as it seems soon. Classical methods are directly or indirectly the foundation of several advanced extraction procedures; therefore, ex- perts mustn’t forget the basics of traditional analytical toxicological sciences.

3. Conclusion

The primary purpose of this research review was to identify the origin and standing of the classification of toxic substances and effective strategies for dealing with their extraction from the autopsy samples in forensic toxicological sciences. This review has recorded several highly appreciable and innovative efforts in these two aspects. The basic extraction procedures have several types and a stretched historical background. Some of these methods have experienced transitional modifications as notified by the literature reports. The advent of the scientific model has also outdone some of these. Few of those method- ologies are still working in the modern era of forensic toxicology. The documentary discussion present in this article is a crucial utility for the medico-legal scientific experts, and the academicians involved in imparting the basic knowledge of toxicological analysis to the future generations. It is also expected that future exploration of these toxi- cology chronicles could be helpful to develop further practical skills.

Declaration of Competing Interest

The author declares that he has no known competing financial in- terests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

It is a matter of immense privilege to thank several online, including a few offline resources used for preparing this article. I acknowledge every single individual whose periodical extract has been made part of this review article. It was a little challenging to select the research pa- pers. Many of these papers now can be considered classical, in that they embody the original work reports of methods. The supplementary ref- erences represented the viewpoints about specific related procedures at various times in field’s history.

References

[1] J.B. Firth, A general account of micro-chemical methods in criminal investigations, J. Crim. Law Criminol. 40 (1949) 381–386 (JSTOR), 〈www.jstor. org/stable/1138558〉.

[2] Casarett and Doull’s Toxicology: The Basic Science of Poisons, 6th ed., C.D Klaassen (Ed.), The McGraw-Hill Companies, Inc., 2001.

[3] A.S. Taylor, Poisons in relation medical jurisprudence and medicine, Philadelphia: Lea & Blanchard, 1848.

[4] R. Saferstein, Criminalistics: An Introduction to Forensic Science, Ninth ed., Pearson Prentice Hall, New Jersey, 2007.

[5] B.S. Finkle, Progress in forensic toxicology: beyond analytical chemistry, J. Anal. Toxicol. 6 (1982) 57–61, https://doi.org/10.1093/jat/6.2.57.

[6] K.D. Watson, Poisoning Crimes and Forensic Toxicology Since the 18th Century Academic Forensic Pathology: The Publication of the National Association of Medical Examiners Foundation, 2020. 〈https://doi.org/10.1177/192536212 0937923〉.

[7] E.G.C. Clarke, in: S. Jickells, A. Negrusz (Eds.), Clarke’s Analytical Forensic Toxicology, Pharmaceutical Press, London, 2008.

[8] S. Bell, Drugs, Poisons, and Chemistry (Essentials of Forensic Science), Facts on File, Inc. New York, 2009.

[9] The Greek Herbal of Dioscorides, Nature 133 (1934) 231–233, https://doi.org/ 10.1038/133231a0.

[10] M.J.B. Orfila, Traité des poisons tirés des règnesminéral, végétal et animal: ou, Toxicologiegénérale, considérée sous les rapports de la physiologie de la pathologie et de la médécinelégale, Vol. 1, A Paris: Chez Crochard, 1818.

[11] A.A. Pappas, N.A. Massoll, D.J. Cannon, Toxicology: Past, Present, and Future, in: Annals of Clinical and Laboratory Science, Vol. 29, Institute for Clinical Science Inc, 1999, p. 254.

[12] M. Flandin, Traite des Poisons, i. 225, ed., 1846 [in A.S. Taylor, 1848]. [13] E. Chapuis, Elements de toxicology, 1873 (Cited Klaassen, 2001). [14] F. Peterson, W.S. Haines, R.W. Webster, Legal Medicine and Toxicology. 2nd ed.

Vol 2. Philadelphia: Saunders& Co, 1923. [15] J.P. Modi, A textbook of Medical Jurisprudence and Toxicology, 26th ed., Kannan

K (Ed.& Revised), LexisNexis, Wadhwa Nagpur India, 2019. [16] F. Bamford, Poisons, Their Isolation and Identification, 2nd ed., Stewart CP,

London, J. & A. Churchill Ltd, 104 Gloucester Place, W.I., 1947. [17] E.G.C. Clarke, Isolation and Identification of Drugs, Pharmaceutical Press,,

London, 1969. [18] L.J. Casarett, J. Doull, Casarett and Doull’s Toxicology: The Basic Science of

Poisons, Macmillan, N.Y, 1975. [19] L.J. Casarett, J. Doull, Casarett and Doull’s Toxicology: The Basic Science of

Poisons, Macmillan, N.Y, 1980. [20] E.G.C. Clarke, in: A.C. Moffat, M.D. Osselton, B. Widdop (Eds.), Clarke’s Analysis

of Drugs and Poisons, 3rd ed., Pharmaceutical Press, London, 2004. [21] M. Essig, Poison murder and expert testimony: doubting the physician in late

nineteenth-century America, Yale J. Law Hum. 14 (2002) 177–210. [22] S. Hempel, The art of medicine James Marsh and the poison panic, Lancet 381

(2013) 2247–2248, https://doi.org/10.1016/S0140-6736(13)61472-5. [23] R. Chatterjee, Toxicology in ancient India, J Indian Academy of Forensic Sci.,

(Proceedings of the symposium on forensic toxicology Hyderabad), 11 (1972) 29–30.

[24] W.R. Cullen, Is Arsenic an Aphrodisiac?. The Sociochemistry of an Element Royal Society of Chemistry, Cambridge, U.K, 2008.

[25] D.M. Jolliffe, A history of the use of arsenicals in man, J. R. Soc. Med 86 (1993) 287–289. PMCID: PMC1294007.

[26] J.S. Haller, Therapeutic mule: the use of arsenic in the nineteenth century materiamedica, Pharm. Hist. 17 (1975) 87–100.

[27] H.C. Semon, An antidote for arsenic, bismuth, and mercury poisoning, Br. Med J. 12 (1924) 662–664, https://doi.org/10.1136/bmj.1.3302.662. PMID: 20771549; PMCID: PMC2304209.

[28] K.A. Winship, Adverse Drug React, Acute, Poisoning Rev., 3 (1984) 129–160. PMID: 6397979.

[29] A. Lykknes, L. Kvittingen, Arsenic: Not So Evil After All?, J Chem Educ., 80 (2003) 497–500. 〈https://ui.adsabs.harvard.edu/link_gateway/2003JChEd 〉.80.497L/doi:10.1021/ed080p497.

[30] J.M. Hyson Jr.,, A history of arsenic in dentistry, J. Calif. Dent. Assoc. 35 (2007) 135–139.

[31] ATSDR. Toxicological Profile for Arsenic. Agency for Toxic Substances and Disease Registry, U.S. Department of Health and Human Services, Atlanta, GA. Available at: 〈http://www.atsdr.cdc.gov/ToxProfiles/tp2.pdf, 2007〉.

[32] P. Bartrip, A ‘Pennurth of Arsenic for rat poison’: the arsenic act, 1851 and the prevention of secret poisoning, Med. Hist. 36 (1992) 53–69, https://doi.org/ 10.1017/S0025727300054624.

[33] J.W. Mellor, A Comprehensive Treatise on Inorganic and Theoretical Chemistry, Vol. IX, Longmans, Green, and Company, London, 1924.

[34] W.A. Campbell, Some landmarks in the history of arsenic testing, Chem. Br. 1 (1965) 198–201.

[35] W.B. Jensen, Notes from the Oesper Collections. The Marsh Test for Arsenic. Museum Notes, May/June: 2014, 1–6. 〈http://hdl.handle.net/2374.UC/731817〉.

[36] S.H. Webster, The development of the Marsh test for forensic, J. Chem. Educ. 24 (1947) 487–490, https://doi.org/10.1021/ed024p487.

[37] Dictionary of National Biography, Oxford University Press, London, Vol. 12: 1100, (1921-22).

[38] J. Emsley, The Elements of Murder: A History of Poison Oxford, Oxford Univ. Press, 2005.

[39] J.C. Whorton, The Arsenic Century: How Victorian Britain Was Poisoned at Home, Work, and Play. esp. Ch. 4: “The Chief Terror of Poisoners, Oxford Univ. Press, 2010.

[40] C.W. Scheele, Om Arsenikochdesssyra (On arsenic and its acid), Kongliga Vetenskaps Academiens Handlingar (Proceedings of the Royal Scientific Academy [of Sweden]), 36, 1775, 263–294.

[41] J.D. Metzger, Handbook of State Medicine Studies, Nathanael Sigismund Frommann’s heirs, printer. Imprint Züllichau: Bey NS Frommanns sel. Erben, 1787. (〈https://lawcat.berkeley.edu/record/214353?ln=en〉).

[42] V. Rose, Ueber das zweckmäßigste Verfahren, um bei Vergiftungenmit Arsenikletzernaufzufinden und darzustellen" (On the most effective method, in cases of poisoning with arsenic, to discover and show the later), J. für Chem. und Phys. 2 (1806) 665–671.

R. Singh

Forensic Science International: Reports 5 (2022) 100275

12

[43] Ueber die Arsenikvergiftung, ihreHülfe und gerichtlicheAusmittelung / von Samuel Hahnemann. 1755-1843. Leipzig:Bey Siegfried LebrechtCrusius, 1786. 〈[https://www.chemeurope.com/en/encyclopedia/Marsh_test.html]〉.

[44] Marsh test. 〈https://www.chemeurope.com/en/encyclopedia/Marsh_test.html〉. [45] Le Droit, 10 June 1840, 5(138):561 (cited in cited in Bertomeu-Sánchez J.R.,

2013). [46] J.R. Bertomeu-Sánchez, Managing Uncertainty in the Academy and the

Courtroom: Normal Arsenic and Nineteenth-Century Toxicology, 2013; Isis 104: 197–225. JSTOR, www.jstor.org/stable/10.1086/670945.Accessed 17 June 2021.

[47] E. Hodgson, A Textbook of Modern Toxicology, third ed., John Wiley & Sons Inc, New Jersey, 2004.

[48] J.R. Bertomeu-Sánchezand A. Nieto-Galan, Chemistry, Medicine, and Crime: Mateu J.B. Orfila (1787–1853) and his times. Watson Publishing International, Science History Publications/usa Sagamore Beach, 2006.

[49] J.R. Bertomeu-Sánchez, From forensic toxicology to biological chemistry: Normal arsenic and the hazards of sensitivity during the nineteenth century”, Endeavour 40 (2016) 82–92, https://doi.org/10.1016/j.endeavour.2016.03.007.

[50] R.E. Gaensslen, Sourcebook in Forensic Serology, Immunology, and Biochemistry; U.S. Dept. of Justice, National Institute of Justice: Washington, 1983.

[51] A. Chevallierand Noticesur Orfila, Ann Hyg Publique Med. Leg., 50 (1853) 450–458. [in International Bibliography of the History of Legal Medicine, JaroslavNemec (Ed), U.S. Department Of Health, Education, And Welfare Public Health Service National Institutes of Health National Library of Medicine, Maryland 20014 DHEW Publication No. (NIH) 73–535. [〈https://www.ojp.gov/ pdffiles1/Digitization/14263NCJRS.pdf〉].

[52] B.B. Beeson, Orfila-Pioneer Toxicologist, Ann Med Hist., 2 (1930) 68–70. PMCID: PMC7945796.

[53] K. Hemanth, M. Tharmavaram, G. Pandey, Chapter 1, History of Forensic Science, in: D. Rawtani, C.M. Hussain (Eds.), Technology in Forensic Science: Sampling, Analysis, Data and Regulations, WILEY-VCH GmbH, Boschstr, 2020, https://doi. org/10.1002/9783527827688 ch1.

[54] J.S. Stas, Nature, 46, 1892, pp. 81–83, https://doi.org/10.1038/046081a0. [55] S.K. Chatterji, An improved method of extracting organic poisons for use in

toxicological analysis, Ph.D. thesis. University of Edinburgh, May 1938.〈https: //era.ed.ac.uk/bitstream/handle/1842/29653/ChatterjiSK_1938redux.pdf? sequence=1&isAllowed=y. http://hdl.handle.net/1842/29653〉.

[56] J. Timmermans, Jean Servais Stas, J Chem Educ., 15 (1938) 353–357. doi: 10.1021/ed015p353.- translated into English by Ralph Oesper.

[57] T. Daldrup, R. Wennig, Proceedings of the 24th TIAFT Conference, Banff, Edmonton, 1987, pp538–542.

[58] D.T. Burns, H. Deelstra, Some aspects of the rise of analytical chemistry in Belgium, Anal. Bioanal. Chem. 391 (2008) 1113–1117, https://doi.org/10.1007/ s00216-007-1777-y.

[59] R. Wennig, Back to the roots of modern analytical toxicology: Jean Servais Stas and the Bocarmé murder case, Drug Test. Anal. 1 (2009) 153–155, https://doi. org/10.1002/dta.32.

[60] R. Wennig, Forensic toxicology, in environmental toxicology and human health, Encycl. Life Support Syst. (EOLSS) (1992). 〈http://www.eolss.net/Sample-Chapte rs/C09/E6-12-23-00.pdf〉.

[61] A. Lacassagne, De L’empoisonnement, Pŕecis De. ḾedecineĹegale. Mass. Cie. (1906) 651–710.

[62] H.G. Fletcher Jr., The history of nicotine, J. Chem. Educ. 18 (1941) 303, https:// doi.org/10.1021/ed018p303.

[63] A.W. Blyth, M.W. Blyth, Poisons: Their Effects and Detection, fourth ed. Charles Griffin and Exeter Street, Strand, London, 1906, pp. 15–16.

[64] J. Wisniak, Jean-Servais Stas Atomic mass of the elements, phlorizin, and toxicology, Rev. Cenic. Cienc. Quím. 50 (2019) 138–154.

[65] F.J. Otto, Manual of the Detection of Poisons by Medico-Chemical analysis translated from the German, with notes and additions by Elderhorst W (ed.), New York: Bailliere, 1857.

[66] F.J. Otto, Anleitungzur Ausmittelung der Gifte. Braunschweig, 1856. [67] U. Pohl, Friedrich Julius Otto (1809-1870): Pharmazeut, Chemiker, Technologe,

Gesundheitsbeamter und das Collegium Carolinum in Braunschweig, in: Braunschw Veroff Gesch Pharm Naturwiss, 40, 1998, pp. 1–413.

[68] B.P. Parker, P.L. Kirk, Toxicological, in: L. Chemistry Meites (Ed.), analysis: In Handbook of Analytical, McGraw-Hill Book Company Inc, USA, 1963.

[69] H. Brandenberger, Dr Alan Stewart Curry, 1925–2007, Forensic Toxicol. 25 (2007) 53–54, https://doi.org/10.1007/s11419-007-0038-2.

[70] A.S. Curry, Toxicological analysis, J. Pharm. Pharmacol. 12 (1960) 321–339, https://doi.org/10.1111/j.2042-7158.1960.tb12672.x.

[71] A.S. Curry, S.E. Phang, A continuous extractor for use in toxicological analysis, J. Pharm. Pharmacol. 12 (1960) 437–441, https://doi.org/10.1111/j.2042- 7158.1960.tb12687.x.

[72] B.W. Meade, B. Wmoop, D.J. Blackmore, S.S. Brown, A.S. Curry, R. Goulding, G. Higgins, H.J.S. Matihew, M.G. Rinsler, Technical Bulletin No. 24. Simple tests to detect poisons, Ann. Clin. Biochem. 9 (1972) 35–46.

[73] A.S. Curry, Poison Detection in Human Organs, Charles C. Thomas, Springfield, Illinois, USA, 1963.

[74] A.S. Curry, Poison Detection in Human Organs, second ed., Charles C Thomas, Springfield, Illinois, 1969.

[75] A.S. Curry, Poison Detection in Human Organs, third ed., Springfield, 1976. [76] R.H. Fox, Personal Communication Cited in Curry AS, 1976, p. 106. [77] H.M. Stevens, V.W. Bunker, The assessment of various Protein Precipitation

Methods for the Extraction of Basic Drugs from tissue, 1976 (then in press,) [cited in Curry AS. Forensic Science, 1976, p. 141, 1976 (then in press,) [cited in Curry AS.

[78] J. Harrison, R. Leggett, D. Lloyd, A. Phipps, B. Scott, Polonium-210 as a poison, J. Radio. Prot. 27 (2007) 17–40, https://doi.org/10.1088/0952-4746/27/1/001. Epub 2007 Mar 6.

[79] K.G. Furton, J. Rein, Trends in techniques for the extraction of drugs and pesticides from biological specimens prior to chromatographic separation and detection, Anal. Chim. Acta 236 (1990) 99–114, https://doi.org/10.1016/S0003- 2670(00)83303-6.

[80] Examples of specific drug assays, Urine Testing for Drugs of Abuse. NIDA Research Monograph, in: R.L. Hawks, C.N. Chiang (Eds.), 73. Rockville, MD: U.S. Department Health and Human Services PHS ADAMHA, 1986, p. 93.

[81] S.R. Hassan, Laboratory investigation of poisoning cases, JIAFC 11 (1972) 84–95. [82] G.E. Turfitt, Recent advances in toxicological analysis, J. Pharm. Pharm. 3 (1951)

321–337, https://doi.org/10.1111/j.2042-7158.1951.tb13071.x. [83] S.K. Niyogi, Methods of separation of drugs from biological materials; a

quantitative evaluation, J. Forensic Med. 17 (1970), 20-41.contd. PMID: 4907048.

[84] C.P. Stewart, A. Stolman, Toxicology: Mechanisms and Analytical Methods, Academic Press, New York, 1960, pp. 1–11.

[85] G. Tully, Forensic science in England & Wales, a commentary, Forensic Sci. Int. 290 (2018) e29–e31, https://doi.org/10.1016/j.forsciint.2018.06.028. Epub 2018 Jul 6. PMID: 30017662.

[86] W. Autenrieth, W.H. Warren, The detection of poisons and strong drugs, P. Blakiston’s Son & Company, 1905.

[87] J.E. Corry, A review. Possible sources of ethanol ante- and post-mortem: its relationship to the biochemistry and microbiology of decomposition, J. Appl. Bacteriol. 44 (1978) 1–56, https://doi.org/10.1111/j.1365-2672.1978.tb00776. x.

[88] C.L. O’Neal, A. Poklis, Postmortem production of ethanol and factors that influence interpretation: a critical review, Am. J. Forensic Med. Pathol. 17 (1996) 8–20, https://doi.org/10.1097/00000433-199603000-00002.

[89] Chapter 4 -Biomedical sampling, in: G. Vas, K. Nagy, K. Vekey (Eds.), Medical Applications of Mass Spectrometry, Elsevier, 2008, https://doi.org/10.1016/ B978-044451980-1.50006-9.

[90] S.L. Tompsett, Extraction procedures in chemical toxicology, Analyst 93 (1968) 740–748, https://doi.org/10.1039/AN9689300740.

[91] A.S. Curry, The isolation and detection of ergometrine in toxicological analysis, J. Pharm. Pharmacol. 11 (1959) 411–414, https://doi.org/10.1111/j.2042- 7158.1959.tb12573.x.

[92] W.R. Erwin, D. Schiller, W.M. Hoskins, Pesticide residue analyses, preassay purification of tissue extracts by wax column, J. Agric. Food Chem. 3 (1955) 676–679, doi.org/10.1021/jf60054a004.

[93] W.M. Hoskins, W.R. Erwin, R. Miskus, W.W. Thornburg, L.N. Werum, Pesticide Residue Analysis, A Polyethylene-Alumina Column for Purification of Tissue Extracts before Analysis, J. Agric. Food Chem. 6 (1958) 914–916, doi.org/ 10.1021/jf60094a006.

[94] Q.W. Zhang, L.G. Lin, W.C. Ye, Techniques for extraction and isolation of natural products: a comprehensive review, Chin. Med. 13 (2018) 20, https://doi.org/ 10.1186/s13020-018-0177-x.

[95] P.L. Kirk, Toxicology (Isolation, identification and determination of poisons), Annu Rev. Med. 6 (1955) 295–310.

[96] H.M. Stevens, A rapid general screening method for drugs in post mortem viscera, 184-93, J. Forensic Sci. Soc. 7 (1967), https://doi.org/10.1016/s0015-7368(67) 70408-9.

[97] A.R. Alha, R.O. Lindfors, Use of acetone in the isolation of organic poisons from biological material, Ann. Med. Exp. Biol. Fenn. 37 (1959), 149-56.

[98] C.G. Daubney, L.C. Nickolls, An investigation into the methods of toxicological analysis of viscera, Analyst 62 (1937) 851–859, https://doi.org/10.1039/ AN9376200851.

[99] P. Valov, Rapid qualitative and quantitative determination of barbiturates from postmortem specimens, Ind Eng, Chem. Anal. Ed. 18 (1946) 456, https://doi.org/ 10.1021/i560155a021.

[100] G.R. Nakamura, Y. Liu, T.T. Noguchi, A method for the separation and determination of neutral compounds in postmortem tissues, J. Anal. Toxicol. 5 (1981) 162–164, https://doi.org/10.1093/jat/5.4.162.

[101] R.J. Ozretich, W.P. Schroeder, Determination of selected neutral priority organic pollutants in marine sediment, tissue and reference materials utilizing bonded- phase sorbents, Anal. Chem. 58 (1986) 2041–2048, https://doi.org/10.1021/ ac00122a025.

[102] J. Azmir, I.S.M. Zaidul, M.M. Rahman, K.M. Sharif, A. Mohamed, F. Sahena, M.H. A. Jahurul, K. Ghafoor, N.A.N. Norulaini, A.K.M. Omar, Techniques for extraction of bioactive compounds from plant materials: A review, J. Food Eng. 117 (2013) 426–436, https://doi.org/10.1016/j.jfoodeng.2013.01.014.

R. Singh

Forensic Science International: Reports 5 (2022) 100275

13

[103] E.G.C. Clarke, Isolation and Identification of Drugs, The Pharmaceutical Press, London, England, 1974, pp. 23–24.

[104] M.D. Osselton, M.D. Hammond, P.J. Twitchett, The extraction and analysis of benzodiazepines in tissues by enzymic digestion and high-performance liquid chromatography, J. Pharm. Pharm. 29 (1977) 460–462, https://doi.org/10.1111/ j.2042-7158.1977.tb11369.x.

[105] R.W. Mayes, The extraction and identification of drugs in aviation toxicology, Aviat. Space Environ. Med. 53 (1982) 332–335.

[106] L.R. Goldbaum, T.J. Domanski, Detection and identification of micrograms of neutral drugs in biological samples, J. Forensic Sci. 11 (1966) 233–242.

[107] P.D. Whitter, L. Paul, A. Cary, A rapid method for the identification of acidic, neutral, and basic drugs in postmortem liver specimens by toxi-lab®, J. Anal. Toxicol. 10 (1986) 68–71, https://doi.org/10.1093/jat/10.2.68.

[108] H. Lord, J. Pawliszyn, Microextraction of drugs, J. Chromatogr. A 902 (2000) 17–63, https://doi.org/10.1016/S0021-9673(00)00836-0.

[109] M. De Boeck, W. Dehaen, J. Tytgat, E. Cuypers, Microextractions in forensic toxicology: the potential role of ionic liquids, TrAC 111 (2019) 73–84, https:// doi.org/10.1016/j.trac.2018.11.036.

[110] R. Jain, R. Singh, Applications of dispersive liquid–liquid micro-extraction in forensic toxicology, TrAC 75 (2016) 227–237, https://doi.org/10.1016/j. trac.2015.07.007.

[111] Y. He, M. Concheiro-Guisan, Microextraction sample preparation techniques in forensic analytical toxicology, Biomed. Chromatogr. BMC 33 (2019), e4444, https://doi.org/10.1002/bmc.4444.

[112] L. Meng, S. Chen, B. Zhu, J. Zhang, Y. Mei, J. Cao, K. Zheng, Application of dispersive liquid-liquid microextraction and GC-MS/MS for the determination of GHB in beverages and hair, J. Chromatogr. B Anal. Technol. Biomed. Life Sci. 1 (1144) (2020), 122058, https://doi.org/10.1016/j.jchromb.2020.122058. Epub 2020 Mar 11.

[113] L. Birk, S.O. Santos, S. Eller, J.O. Merib, T.F. Oliveira, Determinations of new psychoactive substances in biological matrices with focus on microextraction techniques: a review of fundamentals and state-of-the-art extraction methods, Forensic Toxicol. 39 (2021) 350–367, https://doi.org/10.1007/s11419-021- 00582-x.

[114] A. Zgoła-Grześkowiak, T. Grześkowiak, A. Szymański, Comparison of biodegradation of nonylphenolpropoxylates with usage of two different sources of activated sludge, J. Surf. Deterg. 17 (2014) 121–132, https://doi.org/10.1007/ s11743-013-1537-2.

R. Singh