Analysis And Identification Of Diet Aids

Published Date: 02 Nov 2017

Abstract

One of the most popular products obtained over the internet nowadays are diet pills. Customers who purchase these usually have little idea of the contents in them due to them being advertised as naturally sourced ingredients, however most of these contain toxic compounds and cause serious side effects. An unknown diet pill was analysed to identify its main component and why it causes such harmful effects. Appropriate methods of analysis including GC-MS, IR spectroscopy and NMR spectroscopy were undertaken and the main component was found out to be 2,4-dinitrophenol. Quantitative methods were also carried out using internal standards to determine the average mass present in the pill which was 142±6mg (n=3) with its purity being 95.8± 0.6%. Further investigations of limits of detection using full scans and single ion monitoring also determined that the lowest mass detected was 3.6 ng of DNP.

Introduction

Dietary aids

Diet aids are increasingly becoming a 21st century fashion trend. They can be obtained at the local pharmacy, supermarket and health food store. Even countless options are available online. Many are not proved to be effective, some may be dangerous and cause fatality. Whether through added exercise or through metabolism alone, people are turning to substances which assist them in losing the excess fat from their bodies. While some of these substances do prove to have such effect, many of these ‘easy way out’ options are riddled with chemicals that are very poisonous to the body. These diet pills have different effects on the body, from causing headache, nausea and breathlessness to stopping circulation to stroke, seizures and suicidal thoughts. The range of dangerous aids is very wide and starts from simple compounds from ephedra and ephedrine to phenylpropanolamine, phenolpthalein and bumetanide.

Dietary aids are not made to equal standards as prescription drugs and they are sold with narrow evidence on their effectiveness. The US Food and Drug Administration (FDA) has always ear-marked many of these harmful substances and has always tried to step in and halt production of drugs that contain these chemicals. In fact, the FDA has prohibited the sale of diet aids that contain ephedra and other ephedrine-like substance. [1] However, many get to fall under the radar and are affecting more and more people. The lack of effectiveness of these pills is a real issue and manufacturers advertise their revolutionary new drug can change someone’s life in a short space of time but the reality is that the consumer is exposed to high blood pressure, seizures, rapid heartbeat, heart attack or strokes, not to mention the cost of having to buy these drugs on a regular basis. Several studies have proved that ephedra causes heart palpitation, as well as psychiatric and gastrointestinal disorders which thus led to the ban of this substance by the FDA. [2] Phenolphthalein on the other hand, has been described as a suspected carcinogen that can damage or cause alteration to DNA. [3]

Diet aids are usually taken orally as tablets or capsules. Capsules can be hard shelled or soft shelled depending on the contents. Hard shelled capsule is normally used for dry powdered ingredients and soft shelled capsules are for oils or active ingredients that are suspended in oil. Both are made from gelling agents such as polysaccharides or animal gelatine. One may consist of one or more dietary ingredients as well as excipients. Excipients are used as diluents, lubricants, glidants, disintegrants, wetting agents and colouring agents. They are inactive substances that help to hold the active pharmaceutical ingredients stable in the drug. [4] Also, excipients can make the drug taste better (e.g. polysaccharide), makes the tablet easier to swallow, and controls the rate of release of the active compound (e.g. magnesium stearate, carboxymethylcellulose). Moreover, disintegrant is a type of excipient, which expands and dissolves when it is wet and causes a drug to react (e.g. cornstarch, sodium starch glycolate). [5] There are many compounds, which act as excipients and many of them have more than one property. [6]

2,4-Dinitrophenol

Dinitrophenolic compounds are employed in many applications. For example, it is used in agriculture as herbicides, pesticides and fungicides. Although the use of pesticides containing dinitrophilic compounds was forbidden in many countries due to their severe toxicity, they are still used in agriculture. The chemical structures of different dinitrophilic compounds determine their use, and several compounds have more than one use. [14]

2,4-Dinitrophenol (2,4-DNP) is a yellow, crystalline solid with a sweet, musty odour and it is used for the synthesis of dyes, explosives, pesticides and other products. [13] However, it is misused as a diet pill and dietary supplement for body builders. It is volatile with steam and sublimes when heated with caution. It is best soluble in ethyl acetate but also soluble in acetone, chloroform, pyridine, carbon tetrachloride, toluene, benzene, alcohol and other aqueous alkaline solutions. Its salts with alkalis and ammonia are explosives and emit toxic fumes of nitrogen oxides when decomposed. 2,4-DNP is incompatible with heavy metals and their compounds.

Exposure to 2,4-DNP arise by direct contact, ingestion and inhalation, but indirect exposure via the environment for consumers is also possible. DNP is told to be an environmental contaminant by the United States Environmental Protection Agency. Amongst 1400 priority sites that needed cleaning up of industrial waste, 61 of them had DNP contamination. It can pollute the air from burning of certain industrial substances, automobile exhaust and when nitrogen reacts in air along with other atmospheric chemicals. The major site of degeneration is the soil, where microorganisms metabolise DNP. Most dinitrophenolic compounds can be absorbed well through skin, gastrointestinal tract or lung and the main systems prone to toxicity are the hepatic, renal as well as nervous systems. Profuse yellow- tinted perspiration may be observed and it is a sign of DNP poisoning

. [13]

Studies show that a daily dose of 300-400mg for 2 weeks can result in 36-95% increase in an individual’s basal metabolic rate. The primary mechanism of toxicity of 2,4-DNP is thought to be stimulation of oxidative metabolism in cell mitochondria by the uncoupling of oxidative phosphorylation. This is what causing the significant increased in metabolic rate, oxygen consumption and body temperature and leads to the loss of body fat. For these effects, 2,4-DNP is being used as a weight-reduction drug, but the adverse effects, particularly skin lesions and eye cataract and also the dosage problems forbid this application. [13] Early symptoms include hyperthermia, sweating, headache and confusion. Severe exposure may result in restlessness, seizures, coma and death. Although this compound is very toxic with the range of LD50 values 50–71 mg/kg in rats [14], it is difficult to prohibit this chemical from being sold, because it is still employed as an effective pesticide.

Analytical techniques

In the initial planning of how to proceed with this experiment, a literature search was done into the topics to familiarise ourselves with common harmful chemicals in diet pills. This took us into the area of ephedrine, phenytoin and sibutramine among others, all of which come with a long list of side effects and complications in the products. The key information we were looking for was in terms of analysis and which methods would give the best information to use. As with most capsules, the outer casing is made up of gelatine substance while encased in the inner portions is a form of powder or a gel like liquid. This meant we would have to be dealing with either or both solid and liquid samples in our investigation. In analytical chemistry, the suitable methods of analysing solid samples that spring to mind include Infrared (IR) spectroscopy in the form of ATR and also the use of KBr discs. While for liquid samples, Gas chromatography-mass spectrometry stands out as one of the most effective ways.

Gas chromatography-mass spectroscopy

Gas chromatography-mass spectroscopy or GC-MS, a combination of gas chromatography (GC) and mass spectroscopy (MS), is used to analyse and identify the components in a sample. Composed of two major building blocks, GC utilizes a capillary column and allows molecules to elute at different times while travelling through the column. This enables the mass spectrometer downstream to capture, ionize, accelerate, deflect, and detect the ionized molecules separately by breaking each molecule into ionized fragments and detecting them using their mass to charge ratio. Applications of GC-MS include drug detection and it is considered the standard for testing drugs mainly because it can satisfactorily identify constituents in the drug [7].

Gas chromatography-mass spectroscopy/Selected ion monitoring

Operation of a GC/MS in selected ion monitoring (SIM) mode allows for detection of specific analytes with improved sensitivity relative to full scan mode. In SIM mode, the MS gathers data for masses of interest rather than looking for all masses over a wide range. Because the instrument is set to look for only masses of interest it can be specific for a particular analyte of interest. Obviously, the mass spectrum of the analyte must be known to select the masses that distinctively characterise the molecule. This information can be obtained from the reference spectrum by doing a Library Search. [15]

Infrared spectroscopy

IR spectroscopy is widely spread and applied within the pharmaceutical industry where it is used for the analysis of the active substances contained in tablet pills or powdered pills. Simply in IR spectroscopy, the absorption of different IR frequencies is measured with the analysed sample being placed in the IR beam’s direction. Specific functional groups present in the sample can be determined using IR spectroscopy and this technique is capable of achieving spectra from wide range of sample types, solids, liquids and gases. In many cases, some form of sample preparation is required in order to obtain a good quality spectrum. KBr disc method for instance, is one of IR spectroscopy techniques normally used to analyse powdered sample where the sample is ground with dry powdered potassium bromide and pressed into a thin and transparent disc.

ATR on the other hand, does not need sample preparation and it can be employed as to analyse hard gelatine capsules. In ATR, IR beam is focused into a crystal with relatively higher refractive index. An evanescent wave is formed as a result from the IR beam being reflected from the internal surface of the crystal. This is then extended into the sample held in contact with the crystal. When the sample absorbs energy, the evanescent wave is attenuated and this attenuated energy is then passed to the detector. [8] Sample is usually analysed on the single reflection ATR accessories and usually, diamond is preferably chosen because of the advances in the robustness and durability. [9]

NMR spectroscopy

NMR spectroscopy can be employed for further investigation of the pills even though it is intrinsically a technique with low sensitivity because the amount of energy absorbed during the observation of an NMR transition is small due to a small population difference between associated pair of energy levels. However it acts well as a method of product verification as it shows protons and their positions on different carbons. The fact that NMR can identify just about any molecule with little or no sample preparation and analysis means it is a great tool for analysis. It is highly complementary to mass spectrometry where NMR collects information about the functional building blocks that make up a molecule and then offers clues about how those pieces are connected to one another after mass spec has gathered information about it. "A sample is immersed in a strong magnetic field which aligns the magnetic nuclear spins of the atoms in the sample, similar to the way small magnets align themselves when placed near a larger magnet. This alignment is then temporarily perturbed with a radio frequency (RF) pulse, causing the atoms to absorb energy and then radiate it. The exact frequency at which this absorption and emission occurs identifies the kind of atom, provides information about nearby atoms, and indicates how much of each is present. Liquid helium-cooled superconducting magnets are needed to generate the very strong magnetic fields demanded by this technique."[10]

Liquid chromatography-mass spectroscopy

Recently, more methods are being developed for drug analysis and liquid chromatography-mass spectroscopy (LC-MS) is one of the common techniques for pharmaceutical analysis. [11] LC-MS is a brilliant technology for its analysing capability and it is well known for its sensitivity and specificity. [11] It plays an important role in the studies of drug metabolism and analysis, identification and characterization of impurities and degradants in drug contents by providing useful information such as molecular weight and fragmentation patterns for structure elucidation. LC-MS is usually reverse-phase chromatography, where in the presence of hydrophilic solvent such as water, the metabolite binds to the column by hydrophobic interactions and is eluted by a more hydrophobic solvent such as ethyl acetate. [12] These metabolites enter the mass detector by appearing at the end of the column and get ionised by removal of solvents. The detector can work with ions but not neutral molecules and these ions will only pass via good vacuum so solvent removal is very important and a vital step. The ionization technique used depends on the structure and the basicity or acidity of the drug identified. [11] The mass detector then scans the molecules according to mass and gives a complete high-resolution spectrum, separating all ions having different masses. [12]

The purpose of this experiment was to identify and analyse an unknown diet pill which was purchased on the internet.

Experimental

Chemicals

Ethyl acetate (CAS: 141-78-6, analytical reagent grade), acetone (CAS: 67-64-1, analytical reagent grade) and dichloromethane (CAS: 75-09-2, laboratory reagent grade) were purchased from Fisher Scientific UK. 4-nitrophenol (CAS: 100-02-7) was purchased from Sigma-Aldrich and 4-chloro-2-methylphenol (CAS: 1570-64-5) from Lancaster Scientific, England.

Apparatus

Separation and mass detection were done using GC-Perkin Elmer Autosystem XL and MS-Perkin Elmer Turbo Mass MS. The 30 m Themes Restek RXI1-MS column with 250 µm thick stationary phase and 0.25 mm internal diameter was used. The carrier gas was helium and the flow was 1 ml min¯¹. The injection was automatic and the injected volume was 1.0 µl at 200 °C with split ratio being 5:1. The oven program was set to 20 min run, started from 60 °C and heated up to 280 °C (maximum temperature 350 °C).

Single ion monitoring method for limit of detection was set to 3 masses: 107, 154 and 184.

Perkin Elmer FT-IR Spectrometer Spectrum RX1 was used to measure pill contents with KBr method and capsule was analysed using diamond-ATR method.

Oxford NMR AS400 was used for 1H and 13C NMR analysis.

Procedures

The pill was tested by separating pill contents from the gelatine capsule. Several solutions (c=1.0±0.1 mg ml¯¹) were prepared to find out the solubility (methanol, dichloromethane, acetone, ethyl acetate, isopropanol). Ethyl acetate was chosen as the most appropriate. The pill contents did not completely dissolved in any solvent and the remaining particles were filtered.

Internal standard method was used for quantitative analysis. Three internal standards were tried: 4-nitrophenol, 4-chloro-2-methylphenol and 4-methylphenol. Four solutions were made by adding constant volume of DNP (500 µl, c=1.070±0.003 mg ml-1) and varying amount of 4-chloro-2-methylphenol (250±2 µl to 1000±7 µl).

Limits of detection was analysed by preparing seven solutions. They were made in the range from 0.018±0.001 mg ml-1 to 0.18±0.07 mg ml-1 by adding 0.0250±0.0002 ml - 0.250±0.002 ml volume of DNP stock solution (c=1.070±0.003 mg ml-1).

Deuterated solvents were used for NMR experiment. CDCl3 was used to analyse pill contents and D2O was used to dissolve and test the capsule.

Results and discussion

Conclusions

The aim of the investigation was to analyse and identify an unknown diet pill. Appropriate avenues of analysis were investigated to find this to be 2,4-dinitrophenol, a toxic chemical banned from use after the 1930s but is emerging as a diet aid in recent years. the average mass present in the pill was 142±6mg (n=3) with its purity being 95.8 ± 0.6% and LOD using single ion monitoring was 3.6ng of DNP.

Without time limits, further work could be carried out for testing the pill more thoroughly. It would include LC-MS analysis of the pill contents, to find out the chemical components of excipients and also to investigate the hard shell capsule to determine what it is made of. Another method would be voltammetric analysis, where a simple differential pulse will be used to determine the different voltammetric peak potentials of substance mixtures [13]