Sources to the environment easily.2 Fungi, bacteria

Sources and Toxicity of Cyanide
Cyanide Sources
The word “Cyanide” (CN) originated from the Greek word kyanos meaning dark blue. CN can be recognizing in the various spheres of the nature. These CN sources can be categorized into three major sections such as naturally developed, manmade and industrially developing. The natural CN sources are cassava root, yams, maize, bitter almonds; apricot, apple, and peach seeds where CN is present as cyanoglucosides.1 Other than that, cosmos, lightening, volcanic eruption can form CN in to the environment easily.2 Fungi, bacteria and animas itself have abilities to produce CN chemical as their self-defense agent. Manmade CN sources can be listed as follows; cigarette smoke, fires (burning acrylonitrile, wool, silk) and industrial metal complexed smoke can also be considered as the identified CN sources.3 CN is used in electroplating industry,4 gold and silver extraction industry,5 aluminum/nickel battery constituent in battery industry6 which are the examples for the third section of CN sources. Hydrogen cyanide, acidic form of CN, was first isolated from cherry laurel by a Swedish chemist, Karl Wilheim in 1782.7 Since it has the appearance of white crystals, many have misunderstood as white sugar or salt.8 CN is a monovalent combining group which contains the cyano group (C?N). It has molecular weight of 26.018 g/mol and has formal charge of -1 which supportive for complexation.9 In ancient Egypt, CN has been used as one of the toxic compounds for judicial executions (they have identified the higher content of CN in peach seeds),10 and also used as an additive component of gold containing drug developed in ancient Chinese experiments.11 Very recent uses of CN is as a warfare agent; HCN gas used in World War II, by the Nazis against the innocent holocaust12 and used as suicidal agent in war against Sri Lankan government by Liberation Tigers of Tamil Eelam (LTTE) terrorist group.13
Toxicity of CN
CN is a very toxic agent, which is able to take away the cell oxygen utilization of living beings. The possible toxicity may occur through inhalation, dermal absorption, ingestion or parenteral administration.12 Exposure to the CN through one of these may cause severe illness or death. Basically what happens is the cyanide enters to the mitochondria of the living cell and binds to the terminal enzyme of the electron transport chain cytochrome c oxidase resulting in acute cellular hypoxia which prevents the production of ATP inside the cell while destroying the cell.14 Due to these results, the anaerobic pathway turns out to be dominant, and helps to reduce pyruvate to lactic acid through lactic acidosis. This leads to central nervous system (CNS) and myocardial depression.3 Activity of the CN may be depending on the type of the exposure but it is only a matter of time. There are several types of CN poisonings as examples, inhalation of smoke from industrial or residential activities, oral administration through food and drinks and, dermal ingestion threat attack.15 CN can be present as molecular form of hydrogen cyanide (HCN) and anion CN-. HCN is known as a weak acid with a pKa of 9.2, which is stable in the human body pH conditions. Critically, HCN can easily penetrate through the cell membranes and subcellular membranes.3 High doses of CN (>5x LD50) are able to produce more critical responses. For example, induction of pulmonary arteriolar and coronary vasoconstriction, that can cause cardiogenic shock or pulmonary edema. The lower doses of CN result in dizziness, headache, vomiting and nausea due to the inhibition and interfering of cellular enzymes.
Antidotes for the CN Intoxication
Different Types of CN Antidotes
There are several types of CN antidotes have been identified after several years of researches. The two identified mechanisms of actions to treat the CN intoxication are the use of scavengers and detoxification.
This type of antidotes can form stable complexes with CN, such as methemoglobin and cobolt compounds. Amyl nitrite, Sodium nitrite and 4-Dimethylaminopheol can be mentioned as examples for methemoglobin formers. Formation of methemoglobin have shown in following figure (Error! Reference source not found.).

Figure 1. Conversion of Hemoglobin to Methemoglobin.

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The activity of the scavenger type of molecule will be discussed in the later paragraphs. One of the two major antidotes available in US is a methemoglobin former which have used this concept when they develop the antidote.
In the United States currently available major CN antidotes are the NithiodoteTM and the Cyanokit®. NithiodoteTM contains a combination of sodium nitrite and sodium thiosulfate (TS). Sodium nitrite can convert oxyhemoglobin to methemoglobin by changing the oxidation state of the heme center from Fe2+ to Fe3+ (Figure 2). Methemoglobin has the ability to trap the CN- anion in its heme center and forms cyanomethemoglobin. TS converts CN to the less toxic thiocyanate (Figure 3) and excrete form the body with urine.
?NO?_2^- (aq) +2 ?Fe?^(2+) (aq) ?NO?_3^- (aq)+2 ?Fe?^(3+) (aq)
Figure 2. Oxidation State Conversion of Heme Center by Nitrite.

S_2 O_3^(2-) (aq)+ ?CN?^- (aq) ?SCN?^- (aq)+?SO?_3^(2-) (aq)
Figure 3. Conversion of Toxic CN into Less Toxic Thiocyanate.

Cyanokit® contains hydroxocobalamin as the active agent. Hydroxocobalamin can bind CN- to its metal center and convert it to cyanocobalamin (Figure 4). During this process, the body can easily remove CN and reduce its toxic effect.
Both of these antidotes have limitations of the requirement of intravenous (IV) administration that is a practical issue when treating number of victims. In addition, TS has low sulfur donor efficacy which is unfavorable to CN conversion. The presence of endogenous rhodonase (Rh) enzyme, catalyze the conversion of CN by TS. Excessive use of nitrite may lead to production of excess methemaglobin, which can cause methemoglobinemia result in reducing oxygen utilization. Cyanokit® needs in situ preparation and high injection volume (>200 mL).16 Due to the identified following drawbacks, the scientists have been studied possibility of use of easy sulfur donating molecule. The journey of investigating new CN antidotes resulted in three new types of antidotes: a) sulfur donor molecules such as DMTS, b) cobinamide (successor of hydroxocobalamin), and c) sulfanegen (successor of the mercaptopyruvate).17,18 Figure 5 shows the mechanism of sulfur donation.

Figure 4. Hydroxocobalmin Conversion to Cyanocobalamin in the Presence of CN.

?CN?^- (aq)+ S_((Sulfur Donor) ) ?SCN?^- (aq)

Figure 5. CN Conversion to SCN in the Presence of a Sulfur Donor.

Detoxifiers are the molecules which converts CN into less toxic SCN or into different component which easily excrete from the body. Recently understood detoxifiers are listed as TS itself, sulfanegen 3-mercaptopyruate (3-MP), Cystine and ?-ketoglutarate (?-KG).19–21 The CN conversion to SCN by SCN is catalyzed by Rh and 3-mercaptopyruate sulfurtransferase enzymes. Here the both TS and 3-MP act as sulfur donors, which have identified as one of the leading detoxification mechanism. Cystine act as another detoxifier which remove CN form the medium by producing 2-amino-2-thiazoline-4-carboxylic acid. Other than the previously discussed detoxification mechanisms, there is an alternative detoxification process, which CN reacting with ?-KG. Here formation of ?-ketoglutarate cyanohydrin eliminate CN being toxic. This ?-KG is found in animals. Metabolic pathways of CN through detoxifiers have been summarized as below (Figure 6).20

Figure 6. CN Detoxification Pathways.20

Another discovered detoxifier molecule is dimethyl trisulfide (DMTS) which act as a sulfur donor. This molecule also has ability to convert hemoglobin into methemoglobin, which can be considered as a scavenger.16,22
Dimethyl Trisulfide (DMTS)
DMTS is a naturally available compound. It is present in the highest concentration in garlic. It is also used as a flavor enhancer and food additive in the food industry.23 Allicin, which gives the characteristic garlic odor, can be extracted with ethanol. A series of lipophilic molecules are produced by the decomposition of alliin catalyzed the alliinase enzyme in garlic. The various decomposition products are soluble in fats, oils and non-polar solvents, but insoluble in water.16 Alliin can also undergo different reactions to produce various di- and polysulfide compounds. Sulfur containing compounds in garlic, such as DMTS and diallyl disulfide, have been studied as a sulfur donating compounds which can use as CN antidote.24 DMTS is also present in many natural sources such as broccoli, cabbage, cauliflower, aging beer and fungating cancer wounds. The application of DMTS as a CN antidote and its formulation as a CN antidote have been patented by Dr. Petrikovics’ lab (Sam Houston State University, Huntsville, Texas). under the title of CN antidote compositions (US 20150290143 A1, 2015; US 20150297535 A1, 2015).25,26 Unlike TS, DMTS can convert CN to SCN with high efficiency with and without Rh, and has higher antidotal potential than TS. The IM administration makes formulated DMTS superior over the Nithiodote™ and the Cyanokit® as victims can self-administer the antidote during CN exposures.

Figure 7. Structure of the DMTS.

Formulated DMTS
Literature data have shown that DMTS has higher efficiency compared to thiosulfate to donate a sulfur atom to react with CN.22 The middle sulfur atom of DMTS is the proposed one which binds to CN. Dr. Petrikovics’ Lab, Sam Houston State University (SHSU), patented this molecule and its formulation as a CN antidote.26 Several sulfur donor type molecules have been tested and the garlic component DMTS proved to be the best CN antidote. The next step was to develop formulations for intramuscular (IM) administration and to characterize the formulations in vivo and in vitro. One of the DMTS formulations developed by Dr. Petrikovics’ Lab is the 15% polyoxyethylenesorbitan monooleate 80 (poly80). There is three different types of DMTS related formulations have been used in Dr. Petrikovics’s Lab.
Formulation 1 (F1): Lipid-Based Formulation (Micellar and Liposomal Encapsulation)
Formulation 2 (F2): 15% polysorbate 80 (poly 80) – (Patented at Sam Houston State University, Huntsville, Texas)
Formulation 3 (F3): polyoxyethylene sorbitan monolaurate (tween 80) : sorbitan monooleate (span 80) (3:1) – (Developed at Southwest Research Institute (SwRI), San Antonio, Texas)
After developing an efficient DMTS formulation, it needed to be characterized for future use. Stability, size distribution, in vivo efficacy, pharmacokinetics, blood brain barrier penetration and organ distribution experiments were conducted. The major focus of this thesis project was the storage stability investigations.
Lipid-Based Formulated DMTS Click here to enter paragraph text.

15% Poly 80 Formulated DMTS
This formulation was prepared by mixing pure DMTS with 15% aqueous poly 80 solution. The solubility of DMTS in water is only 0.3 mg/mL, therefore it was necessary to enhance this feature. The surfactant poly 80 helped to dissolve DMTS in water up to 100 mg/mL concentration. Former member of this research group Dr. Lorand Kiss compared the storage stability of the 15% poly 80 formulated DMTS in crimped sealed vials versus ampules.
Tween 80 : Span 80 (3:1) Formulated DMTS
This formulation was developed in Southwest Research Institute, San Antonio, Texas. The initial goal of formulating this formula is to overcome the low stability of F1 and high injection volume requirement of F2. This formulation was prepared using tween 80 and span 80 in a ratio of 3:1. Then DMTS was added to it until amount of DMTS is 40% (m/m). When this solution is prepared the final concentration of the F3 formulation would be 400 mg/mL. Therefore requirement of high injection volume can overcome with this formulation. F3-formulation still needs to be characterized for its future use. Recent studies have found that slower absorption rate as its one of the disadvantage

Oxidation of DMTS
It has been reported that dialkyl polysufides undergo disproportionation reactions.24 The reaction rate depends on the temperature, especially at higher temperatures such as 145 – 160 ºC. The thermally decomposed products are also thermally unstable and they undergo further decomposition.24 Through reduction reactions, thiols and hydrogen sulfide are formed, while when oxidation reactitons take place, thiosulfate and thiosulfonate are formed from the organic polysulfides.25

Analytical Techniques Used for Analysis
Gas Chromatography – Mass Spectrometry (GC-MS)
GC-MS is a combined instrumentation that allows for qualitative analysis of complex solutions. GC-MS is indicated as one of the primary methods available for qualitatively identifying the molecular makeup of a sample. GC divides molecules based on their chemical properties about an internal column gas affinity. MS fragments components, ionizing them and then separating these fragments based on their mass-to-charge ratio (M/Z). To generalize the process, GC separates elements of a complex sample and MS provides details of these individual components lending to their identification.
Gas Inlets
Gas is fed from cylinders through supply piping to the instrument. It is usual to filter gases to ensure high gas purity and the gas supply may be regulated at the bench to ensure an appropriate supply pressure. Carrier – (H2, He, N2) Make-up gas – (H2, He, N2) Detector Fuel Gas – (H2 & Air, Ar or Ar & CH4, N2) depending on the detector type.
Pneumatic controls
The gas supply is regulated to the correct pressure (or flow) and then fed to the required part of the instrument. Control is usually required to regulate the gas coming into the instrument and then to supply the various parts of the instrument. A GC fitted with a Split/Splitless inlet, capillary GC column and Flame Ionization detector may have the following different gas specifications: Carrier gas supply pressure, column inlet pressure (column carrier gas flow), inlet split flow, inlet septum purge flow, detector air flow, detector hydrogen flow, detector make-up gas flow. Modern GC instruments have Electronic Pneumatic pressure controllers – older instruments may have manual pressure control via regulators.
Here the sample is volatilized and the resulting gas entrained into the carrier stream entering the GC column. There are many inlet types exist including Split / Splitless, Programmed Thermal Vaporizing (PTV),Cool-on-column (COC) etc. The COC injector introduces the sample into the column as a liquid to avoid thermal decomposition or improve quantitative accuracy.
In GC, retention of analyte molecules occurs due to stronger interactions with the stationary phase than the mobile phase. This is unique in GC and, therefore, interactions between the stationary phase and analyte are of great importance. The interaction types can be divided into three broad categories: Dispersive, Dipole, Hydrogen bonding The sample is separated into its constituent components in the column. Columns vary in length and internal diameter depending on the application type and can be either packed or capillary. Packed columns (typical dimension 1.5 m x 4 mm) are packed with a solid support coated with immobilized liquid stationary phase material (GLC). Capillary columns (typical dimension 30 m x 0.32 mm x 0.1 mm film thickness) are long hollow silica tubes with the inside wall of the column coated with immobilized liquid stationary phase material of various film thickness. Many different stationary phase chemistries are available to suit a host of applications. Columns may also contain solid stationary phase particles (GSC) for particular application types.
Column Oven
Temperature in GC is controlled via a heated oven. The oven heats rapidly to give excellent thermal control. The oven is cooled using a fan and vent arrangement usually at the rear of the oven. A hanger or cage is usually included to support the GC column and to prevent it touching the oven walls as this can damage the column. The injector and detector connections are also contained in the GC oven. For Isothermal operation, the GC is held at a steady temperature during the analysis. In, temperature programmed GC the oven temperature is increased according to the temperature program during the analysis.
The detector responds to a physicochemical property of the analyte, amplifies this response and generates an electronic signal for the data system to produce a chromatogram. Many different detector types exist and the choice is based mainly on application, analyte chemistry and required sensitivity – also on whether quantitative or qualitative data is required. Detector choices include: Flame Ionization (FID), Electron Capture (ECD), Flame Photometric (FPD), Nitrogen Phosphorous (NPD), Thermal Conductivity (TCD) and Mass Spectrometer (MS).
Data System.
The data system receives the analogue signal from the detector and digitizes it to form the record of the chromatographic separation known as the ‘Chromatogram’. The data system can also be used to perform various quantitative and qualitative operations on the chromatogram – assisting with sample identification and quantitation.

Figure 8. Schematic Diagram of GC-MS. Click here to enter an explanatory note for your figure, or delete this text if your figure does not require a note.

High Performance Liquid Chromatography (HPLC).
High performance liquid chromatography (HPLC) is a highly improved form of column liquid chromatography. Other than a solvent being allowed to drip through a column under gravity, it is forced through under high pressures of up to 400 atmospheres, which makes it much faster. All chromatographic separations, including HPLC operate under the same basic principle; separation of a sample into its constituent parts because of the difference in the relative affinities of different molecules for the mobile phase and the stationary phase used in the separation. There are several types of HPLCs, which listed below.

Figure 9. Schematic Diagram of HPLC. Click here to enter an explanatory note for your figure, or delete this text if your figure does not require a note.
As shown in the schematic diagram (Figure), HPLC instrumentation includes major components of a pump, injector, column, detector and integrator or acquisition and display system. The heart of the system is the column where separation occurs.
Solvent Reservoir
Mobile phase contents are contained in a glass reservoir. The mobile phase, or solvent, in HPLC is usually a mixture of polar and non-polar liquid components whose respective concentrations can be varied depending on the composition of the sample.
High Pressure Pump
A pump aspirates the mobile phase from the solvent reservoir and forces it through the system’s column and detector. Depending on several factors including column dimensions, particle size of the stationary phase, the flow rate and composition of the mobile phase, operating pressures of up to 42000 kPa (about 6000 psi) can be generated.
Sample Injector.
The injector can be a single injection or an automated injection system. An injector for an HPLC system should provide injection of the liquid sample within the range of 0.1-100 mL of volume with high reproducibility and under high pressure (up to 4000 psi).
Columns are usually made of polished stainless steel, are between 50 and 300 mm long and have an internal diameter of between 2 and 5 mm. They are commonly filled with a stationary phase with a particle size of 3–10 µm. Columns with internal diameters of less than 2 mm are often referred to as microborer columns. The temperature of the mobile phase and the column should be kept constant during an analysis.
The HPLC detector, located at the end of the column detect the analytes as they elute from the chromatographic column. Commonly used detectors are UV-spectroscopy, fluorescence, mass-spectrometric and electrochemical detectors.
Data Acquisition and Analysis
Signals from the detector may be collected on chart recorders or electronic integrators that vary in complexity and in their ability to process, store and reprocess chromatographic data. The computer integrates the response of the detector to each component and places it into a chromatograph that is easy to read and interpret.
Thin Layer Chromatography (TLC)
In thin layer chromatography, a solid phase, the adsorbent, is coated onto a solid support as a thin layer (about 0.25 mm thick). In many cases, a small amount of a binder such as plaster of Paris is mixed with the absorbent to facilitate the coating. Many different solid supports are employed, including thin sheets of glass, plastic, and aluminum. The mixture (A plus B) to be separated is dissolved in a solvent and the resulting solution is spotted onto the thin layer plate near the bottom. A solvent, or mixture of solvents, called the eluant, is allowed to flow up the plate by capillary action. At all times, the solid will adsorb a certain fraction of each component of the mixture and the remainder will be in solution. Any one molecule will spend part of the time sitting still on the adsorbent with the remainder moving up the plate with the solvent. A substance that is strongly adsorbed (say, A) will have a greater fraction of its molecules adsorbed at any one time, and thus any one molecule of A will spend more time sitting still and less time moving. In contrast, a weakly adsorbed substance (B) will have a smaller fraction of its molecules adsorbed at any one time, and hence any one molecule of B will spend less time sitting and more time moving. Thus, the weaklier a substance is adsorbed, the farther up the plate it will move. The more strongly a substance is adsorbed, the closer it will stays near the origin. Several factors determine the efficiency of a chromatographic separation. The adsorbent should show a maximum of selectivity toward the substances being separated so that the differences in rate of elution will be large. For the separation of any given mixture, some adsorbents may be too strongly adsorbing or too weakly adsorbing. Table 1 lists a number of adsorbents in order of adsorptive power.

Figure 10. Setup of the Analysis on TLC Plate. Click here to enter an explanatory note for your figure, or delete this text if your figure does not require a note.


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