Quantification of Opiates in Hair Samples

The analysis of controlled drug abuse has been primarily been carried out using urine samples. This is then complemented further with use of other biological fluids such as blood, sweat and saliva. There are many advantages as to why urine is a better biological fluid to use in comparison to the other biological fluids. Some these include:

• Less invasive – No needles required to obtain the sample.
• Little medical supervision required
• Cost of conducting the test is lower

Urinalysis however also has some limitations, some which are

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• The drug detection window (DDW), the time frame in which the drug can be detected is somewhat short. It is typically 1-3 days.
• The sample can be easily contaminated; therefore testing is carried out on an altered sample.
• The sample can be easily changed if it is diluted. [2]
• There are also safety issues, if improper care is taken when handling the sample then sin contact may lead to infections.
• Abstinence can also provide inaccurate readings. If prior knowledge of when the test is going to be carried out, the user may abstain from using the controlled substance a few day prior to the test being carried out.
• Consumption of excessive water – the user excessive amount of water are consumed then the sample given may be diluted, therefore providing an inaccurate concentration of the drug abuse.
• The cut off point- Urinalysis tends not to have a low enough of a cut off point. This will mistake controlled drug abuse with other possible metabolites of food. An example of this is the consumption of poppy seeds. This can be mistaken for morphine abuse.

With further developments analytical techniques such as GC/MS hair samples are now the preferred sample to for the analysis of controlled drug abuse. This is then complemented with urinalysis and blood analysis. The advantages using hair include

Drugs are commonly found in hair samples.

Hair tends to be more of a qualitative test rather than a quantitative. It measures the concentration and frequency of the abuse, not just its presence. [3]

A longer DDW – measures the abuse the abuse over months and years, as appose to days.

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The chart above shows the concentration of a drug present urine and hair over a period of 12 days

Hair is easier to handle – poses no threat of infection if skin contact is made [4].

Hair is a more stable specimen – it has a stable protein structure which cannot be easily contaminated [5]

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Little medical supervision or surgical intervention is required when obtaining the sample and is therefore seen to be less invasive.

1 – HAIR

1.1 Anatomy of Hair

Hair is made up of two distinct compartments, the shaft and the follicle/root. The shaft is the visible outer part, which comes out of the skin on the scalp. This part is often referred to as the ‘dead’ part of hair. The reason for this is that the compartment within the skin, the follicle has a bulb shaped ending. Within the centre of the bulb there are cells, which are constantly dividing. As new cells are produced the older one are pushed up. As they are moving up they die slowly, which then form the hard shaft.

Each strand of hair is made up of protein fibres called keratins. The chemical composition of keratin includes long chains of amino acids. One key example of these amino acids is cysteine, which contains sulphur. One key ability of sulphur is its ability to form bonds with other sulphur molecules, disulpher bridges. This is type of chemistry is present in hair. Adjacent keratin proteins link together to form disulphide bonds. The molecular interactions between these bonds are quite strong, and therefore it is quite hard to break the bond between them. The disulphide bonds can be broken using an alkali solution, as acidic solutions generally have no effect.

Each strand consists of three layers the cuticle, cortex and medulla. The medulla is made up of cells that are quite large and hollow. The middle layer is the cortex, which makes up the majority of hair. The cells in this layer are tightly packed due to cross links between the keratin chains. The characteristic of hair are predominantly determined by this layer. This includes the colour of hair. Other characteristic determined by the cortex are the flexibility and strength of hair and the also the texture.

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The cuticle is the outermost layer and is formed by a single layer of overlapping tightly packed cells, which are transparent in appearance. This layer provides protection for the cortex and the medulla. This layer also characterises the strength of hair, as its strenuousness it is able to take the effects of any impact.

Within the root is the follicle, this is a multilayered bulb like structure. Where each layer has its own function. At the base of the bulb is the dermal papilla. This is fed by small blood vessels. The function of these vessels is to provide essential nutrients and oxygen to the growing hair, it also removes any waste products. This is also the site of where signals are received, instructing hair to grow. [7] This is done by the presence of hormones and adrenogens. The adrenogens determine when hair grows and also the size of the follicle. Therefore influencing the physical properties of hairs, i.e. thickness.

The hair follicle is covered by two sheaths, the inner and outer. The function of these sheath is to provide protection to the hair shaft. The inner sheath coats the follicle up to the opening of the sebaceous gland. The outer sheath coats the follicle all the way up to the gland.

1.2 The Hair Cycle

Each strand of hair grows in a repeated three stage cycle, starting with the Anagen phase continuing to the Catagen phase and concluding with the Telogen phase. [8]

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Anagen phase: Hair growth phase

Occurs with 85 % of hairs at any one time

Duration: 2-6 Years

Activity: Stimulus received at dermal papilla → Rapid reproduction of keratinocytes within the bulb → Upward movement of keratinocytes

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→ Formation of hair shaft

Catagen Phase: Regression phase

Duration: 1-2 Weeks

Activity: Mitosis cycle ends and reabsorption occurs → The old keratinocytes cells are then preceded by the new ones → Death of the previous keratinocytes → Hair follicle size reduced by 1/6 → Degradation of the lower part → Hair follicle becomes detached → Keratinocytes cells attached to the follicle and the dermal enter the next phase.

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Telogen Phase: Rest phase

Occurs with 15% of hairs

Duration: 3 Months

Activity: Dermal papilla is in a resting state → Towards the end of the stage, the hair and follicle become detached from one another → a new connection made between the lower part of the follicle and the dermal papilla → Once the connection has been made, the cycle will start again → Anagen phase → Formation of a new hair, if the previous hair is still attached to the follicle then the new one will push out the old hair [8]

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1.4 Integration of Opiates Into Hair

There have been several studies carried out that look into how drugs and their metabolites are integrated into hair. These studies have proposed some simple mechanisms as to how this is done. However an in depth explanation would require further studies to be carried out.

As hair has a protein structure, it is able to trap the metabolites present in the blood into hair whilst the structure is being synthesised. This is auctioned whilst the hair is attached to the follicle, i.e. whilst the hair is growing. As the hair fibre is being formed, the drug and its metabolites become integrated. This will result in the drug and its metabolites to be stabilised within the keratin structure.

The basic model proposes a mechanism that a drug and it metabolites may be integrated by passive diffusion. This is where the drug is passively diffused from the dermal capillaries into the growing hair cells. The point at which this passive diffusion occurs is when the hair is follicle length is at a length of approximately 1.2 – 1.5 mm long. This is the length between the hair matrix area and the area of the keratinised area. This suggests that if the hair is 1.2 – 1.5 mm long then drug exposure of about three days is available for analysis.

A more detailed model can also explain how drugs and their metabolites can be integrated into hair. This takes into account different mechanisms occurring various times of the hair growth cycle, and at a number of different locations. The research into this multi-compartment theory was initiated by Henderson [10], but has since been backed up by further studies.

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An example of this is, the movement of the drugs and its metabolites from sweat and Sebum. The integration of the drugs and their metabolites occurs once the hair has been formed. Studies have shown that the concentration of drugs in sweat is higher than the concentration found in blood. This would therefore explain the high concentrations if drugs found in hair. [10] Drugs can also be integrated into hair from the external environment, i.e. from air, water and hair treatments such as hair dying, and perming.

As well as the external environment drugs can also be integrated with hair by intradermal transfer. This happens deep within the skin compartment, where highly lipid soluble drugs can penetrate into the skin layer and then become integrated into hair. [11] Also melanin content may have some influence on the drug being transferred. The drug may associate with melanin sites that are present in the skin. This will result in the transfer of the drug and its metabolites as well as melanin pigment molecule.

The actual properties of the drug being integrated will also ultimately influence which mechanism is used for the drug to be integrated with hair. Examples of these properties include the structural, chemical, and physical properties. When looking specifically at the structural properties, there are three factors that will influence the mechanism undertaken to integrate the hair. [13] These include:

• The melanin content of the hair
• The lipophilicity of the drug
• The basicity of the drug

The influence of melanin on the integration of the drug with has been examined in several studies. A sample of grey hair was analysed. It can be seen that the sample contains white hair and pigmented hair. It was found that even though the root had been placed under the same conditions, i.e. the same concentration of the drug and its metabolites in the blood the pigmented had ten times the concentration of the basic drug compared to the hair sample that was not pigmented. [14] This study carried out by M. Rothe et al prompted further studies to be carried out. These looked at the difference in drug concentration between black, brown, blonde and red coloured hair. The results obtained from this study also found the correlating results.

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The integration of uncharged organic i.e. lipophilic drugs can infiltrate the membrane with ease, as well as being able to diffuse along the concentration gradient. This however is not the case with lipophobic or charged drugs. When they try to infiltrate the membrane a drug resistant barrier is formed, therefore restricting the drug from entering the membrane. Also basic and acid drugs, are highly ionised can enter the membrane if the charge they have is neutralised. This is achieved by deprotonation or protonation. This suggests that the pkA of the drug is an important factor, when it is trying to enter the melanocyte cells so it can be integrated with hair.

Studies have also found that the intracellular pH of melanocytes typically ranges from 3 to about 5. Due to this chemical property, there is an increase in the accumulation of drugs at pigmented sites. However this is not the case for all acidic drugs, so this is why they are often found in lower concentrations. [13]

2. Opiates and Opioids

2.1 Derivation and active component

The opiates are derived from opium. Opium is released from immature seeds that grow within the poppy plant, also known as papaverus somniferum.

The active component from which the opiates are synthesised, is known as the latex. This is a white milk like emulsion fluid that is released, when an incision made on the green wall of the poppy plant seed. The latex is removed typically between 1-3 weeks after the poppy plant has flowered.

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The white latex is then dried, leading to the formation of brown coloured opium. They are a group of about twenty opiate alkaloids. An opiates can however is a synthetic chemical/drug that can be synthesised using an opiate as starting material, or be fully synthesised to mimic the action of an opiate. Morphine is the most prominent opiate present within opium, making up 10%. Codeine is second, which makes up approximately 5% of opium. The other main constituents of opium include thebaine, noscapine and papaverine.

Some of the twenty alkaloids can be synthesised further in laboratories They can be synthesised using an opiate as starting material, or be fully synthesised to mimic the action of an opiate.

An example of this is the synthesis of heroin from morphine. There are also opioids that can be synthesised fully in a laboratory. An example of this type of opioid is methadone.

2.2 Classification

The opiates can be classified into three main categories, natural opiates, semi synthetic opioids and fully synthetic opioids.

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2.2.1 Natural Opiates

These are chemical/drugs that are synthesised directly from the latex that is produced from the seedlings of the poppy plant. Once the latex has been dried it is now known as opium. The natural opiates are then extracted from the dried opium. The most abundant chemical/drug present in the opium is morphine, accounting for 10 % of opium. The second most abundant natural opiate is codeine, account for approximately 5 % of opium. Thebaine is the third most abundant opiate, accounting for approximately 3% of opium.

Thebaine

The chemical composition of morphine and codeine is quite complex. This is why it is not feasible to synthesise them in a laboratory. This therefore means that the best method of obtaining these opiates is through direct extraction from the poppy plant.

2.2.2 Semi Synthetic Opiates

These types of opiates are synthesised using the natural opiates, such morphine as starting points. There are a vast amount of semi synthetic opiates. One example of a natural opiate being used to synthesise a semi synthetic opiate is the production of heroin from morphine.

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The reaction of morphine with acetic anhydride results in the formation of diacytylmorphine, also known as heroin. Morphine as well as the other natural opiates are the starting material for many semi synthetic opiates. The table below shows examples of these semi synthetic opiates.

Semi Synthetic Opiate

Also Known As

Starting Natural Opiate

Chemical Structure

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Hydromorphone

Dihydromorphinone and Dimorphone

Morphine –

Hydrogenated ketone

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Hydrocodone

Dihydrocodeinone

Codeine and Thebaine

Oxycodone

Dihydrohydroxycodeinone

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Thebaine –

The structure is similar to codeine, but differs in 3 ways

1 -hydroxyl group at C-14, codeine has a H

2- has a dihydro between C 7,8, codeine has double C bond

3- carbonyl group present instead of a hydroxyl group

Oxymorphone

14-Hydroxydihydromorphinone

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Thebaine or Morphine – Esterification of the hydroxyl groups

Desomorphine

Dihydrodesoxymorphine

Morphine –

Remove 6-hydroxy group

Saturation of the 7,8 C double bond

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Heroin

Diacetylmorphine

Morphine-

Addition of acetyl ester groups at C 3,6, therefore diacetyl ester of morphine

Codethyline

Ethylmorphine

Codeine or Morphine

the OC2H5 group substituted for an aromatic 3-OH

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2.2.3 Fully Synthetic Opiates

The fully synthetic opioids are completely chemically different to opiates, however the mode of action on the body. The fully synthetic opiates are able to mimic the way morphine acts on the body. The first type of fully synthetic opiates that was synthesised was called meperidine. This was then with the production of methadone.

Some other examples of fully synthetic opiates are fentanyl, pethidine, tramadol and dextropropoxyphene. The advantages of synthesising these synthetic opiates are that the potency of the chemical/drug can be rapidly increased, in comparison to that of morphine.

2.2.4 Endogenous Opiates

These are natural substances that are produced within the brain. The characteristics of the endogenous opiates are similar to that of the alkaloid opiates that derived from the poppy plant, commonly known as exogenous opiates. The endogenous opiates interact with opiates receptors in the same way as the exogenous opiates i.e. causing analgesia and euphoria. Examples of these endogenous opiates are

• Endorphins
• Enkephalins
• Dynorphins

2.3 Mode of Action of Opiates

Opiates are chemicals that act on the body in two ways. The first is by reducing or stopping chemical signals, therefore having sedative effects. This will result in a reduction reaction time in which the body reacts to pain, also helps to decrease the awareness of pain and finally helps increase the tolerance of pain. The second way in which the opiates act within the body is to create a feeling of elation.

The mechanisms that allow the opiates to behave this way is achieved by the interactions that occur at the opiate receptors. The opiate receptors are located mainly in the central nervous system, i.e. the brain and spinal cord and also within the respiratory centre. The body also produces it own natural opiates, known as endogenous opiates. Some examples of these endogenous opiates are endorphins, enkephalins and dynorphins. They are all released naturally to interact with the opiate receptors. The endorphins are located in the hypothalamus, and are released in response to stress. The enkephalins are present within the central nervous system, and act on the pain pathways. The dynorphins are also located in central nervous system, the spinal cord. They are also associated with the pain pathways.

These natural opiates interact with three main opiate receptors mu, kappa and delta, which are g-protein coupled.

The opiates that are derivative of the poppy plant are called exogenous opiates. They also interact with the mu, kappa and opiate receptors. If the use of the exogenous opiate s is abused, adverse effects. As well as the opiates being able to block pain, they also make the user feel elated. This is the result of the opiates reacting with mu opiate receptors. The same receptor that the endogenous opiates, endorphin reacts with. Due to these properties it is often the case that opiates are used recreationally as appose to medically.

3. Extraction of Opiates from Hair

3.1

In order to determine the presence of in a hair sample, the drugs need to be extracted from the hair structure. The reason for this is that there have not been any developments in analytical techniques that analyse the hair and drug when they are combined in one structure. This is why extraction steps are taken to analyse the drug separately from the hair structure. The choice of solvent used for the extraction process must take into consideration the chemical structure of the drug, and what response they will have to the solvent.

3.2 Division of hair in to segments

Hair must be divided into segments prior to the opiates being extracted. As hair grows at a rate 0.5 inches per month [ref -see notes], it provides a timeline of when and at what concentration the opiates we consumed. The hair sample must be all be of the same length prior to being analysed. It is quite difficult to quantify at which period of time the opiate was consumed if a clump of hair is used as appose to a single strand of hair. it is generally get harder the longer the distance from the root. This is why it is beneficial to analyse the hair sample in sections. [25-22]

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Studies carried out, have found the following divisions of a 45 cm length provide the optimum analysis. Staring from the root the following divisions are made:

4 x 0.5 cm

3 x 1.0 cm

2 x 2.0 cm

2 x 3.0 cm

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2 x 5.0 cm

2 x 10 cm

3.3 Decontamination of hair

Prior to any extraction techniques being carried out on hair, any external contaminants must be removed. Although the analytical techniques analyse the opiates that are incorporated within the hair structure, sometimes other substances may be detected if the decontamination process is not actioned correctly. The results of the analysis may account for surface contaminants that may have made contact with hair, i.e. if the user has touched a substance and subsequently touches their hair. This will result in a positive result eng obtained even though the user has not consumed the substance.

Other possible sources of these contaminants may be from hair care products such as shampoos and conditioners. Also any hair styling products, such waxes and hair sprays also need to be removed. As well these sweat and any fatty sebum released from the sebaceous glands need to be removed. Also environmental contaminants such dust need to be discarded. If the sample prior to being cut was exposed to any drugs in the environment, this step will remove this source of contamination. The reason for decontaminating the hair sample is to prevent any background noise when the sample is analysed. The choice of the decontaminant has to have specific properties. This is because it has to remove any external contaminants, however not be able to remove any of the drugs and its metabolites from the hair sample. [15]

Non protic solvents such as dichlormomethane and acetone are good decontaminates as they do not swell the hair, so extraction will not occur.

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Using a 300mg sample of hair is used. It is placed into an ultrasonic bath,

There are series wash cycles performed on the hair sample, and are usually initiated with two washes with dichloromethane. A typical experiment conducted in [16], which tried to determine the opiate content in hair carried out four different wash cycle, on four different samples of hair.

20 ml of dichloromethane, 15 ml of acetone, 15 ml of methanol, 10 ml of methanol.

20 ml of isopropanol, 15 ml of acetone, 15 ml of methanol, 10 ml of methanol.

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20 ml of dichloromethane, 15 ml of isopropanol, 15 ml of methanol, 10 ml of methanol.

20 ml of n-hexane , 15 ml of acetone, 15 ml of methanol, 10 ml of methanol.

This experiment showed that a mixture of solvents could be used to wash the hair samples.

3.4 Disintegration of opiates from hair structure

As there are currently no analytical techniques that test for opiates whilst they are integrated within the hair structure. This means that the hair structure must first of all be digested and then the drugs and its metabolites are extracted, to determine which drugs are present.

There are various solvents used to extract opiates and its metabolites from hair.

3.4.1 Extraction with Methanol

Methanol is a good solvent used to extract opiates from hair. It is hydrophilic, so it can enter the hair structure quite easily. The action of methanol is that it causes the hair to swell up. This will result in the drugs integrated within the hair structure to be released. This is done by the opiates diffusing out. This extraction is carried out in an ultrasonic bath. This helps to degrade the hair structure. There are some impurities still present once this methanol extraction has been carried out. So a secondary clean up is still required. [17]

There are some disadvantages to this extraction method. This is because the amount of drug obtained from the extraction procedure, is quantitatively less than other extraction methods used to derive opiates hair from hair.

However the main disadvantage of using is that using methanol extraction, this is that the opiate extracted can sometimes be hydrolysed. An example is the conversion of 6-monoacetylmprphine (Heroin) to morphine. This results in the non detection of monoacetylmprphine (heroin). [13] Therefore when trying to detect the opiate Heroin, it can be hydrolysed to morphine. Therefore resulting in the heroin present in hair to go undetected. [21]

3.4.2 Extraction with a buffer solution

This extraction procedure is widely used to extract opiates and their metabolites from hair. It generally seen to be one the more successful methods. A typical buffer solution would be a phosphate buffer, at a pH of approximately 6.4 – 7.6. [18] In comparison to methanol phosphate buffer are seen to be a cleaner approach of extracting opiates. In addition to use the phosphate sometimes additional enzyme are added to help to determine intricate metabolites. A typical enzymes used are combination of glucuronidase and arylsulphitase.

3.4.3 Supercritical Fluid Extraction

This method uses a supercritical fluids such carbon dioxide (CO2) to extract opiates from hair.

It is seen to be advantageous over other extraction methods, as supercritical fluids have specific properties that allow them to be more efficient in extracting opiates and their metabolites from hair samples. Some examples of these properties include that physically, supercritical fluids are less viscous than other solvents. This in turn allows them to move more freely. [19]

They have an increased speed of extraction, in particular with opiates. Research carried out into the extraction of opiates from hair using supercritical fluids by Edder et al. It was found that use of the supercritical fluid carbon dioxide, not only speeded up the extraction process but also retrieved a high yield. It was found that 100 % of the morphine that was present in the hair sample was extracted, along with 98.2% codeine, and 92% of methadone. This was all done in a 25 minute procedure. [20]

Other advantages of using supercritical fluids to extract opiates from hair samples are that it has been found that supercritical fluids tend not to contaminate the samples, in comparison to solid phase extraction and liquid liquid extraction. The efficiency of this method also allows the procedure to be more automated in comparison to other extraction techniques.

3.4.4 Enzymatic Digestion of the Hair matrix

This method primarily uses the enzymes pronase and protein kinase A to break down the hair structure. The procedure requires the hair sample to be placed into the enzyme mixture at temperature between 40 -60oc for approximately 6 – 12 hours. [22]

The action of these enzymes is to breakdown the disulphide bonds that are present within the hair structure. Often dithiothreitol is used to aid pronase and protein kinase A, by decreasing the time taken to extract the opiates and their metabolites from the hair sample.

Other enzymes used to breakdown the hair structure include glucuronidase and arylsulphitase.

The disadvantage of using this method in comparison to other extraction techniques is that some of the sample may be altered prior to them being for the analytical tests. An example is the antibodies that are required for radio immunoassays, may be denatured by excessive heating required by this extraction process.

3.4.5 Digestion with Sodium Hydroxide.

The use of alkaline solutions such as sodium hydroxide in digesting hair for the extraction of opiates has proven to be very compatible. This is because unlike acid solvents the constituents of the opiates are not hydrolysed along with the hair structure. An example of study conducted by Aldo Polettini et al found that in some case hair samples of heroin users, when digested in methanol hydrolysed the heroin to morphine. Whereas the hair sample that was digested in sodium hydroxide successfully hydrolysed the hair structure but did not alter the opiate and its constituents. [ref]

Typical experiments digest hair samples in a 2M concentration of sodium hydroxide, set at a temperature of about 79°c for 60minutes.

4. Gas Chromatography / Mass Spectrometry Analysis of Hair Samples

4.1 How GC/MS works

Gas Chromatography and Mass Spectrometry are two separate analytical techniques that are used together to quantitatively detect low concentrations of opiates. This analytical technique has exceptional specificity to the detecting in opiates in hair, with levels ranging from nanograms to picograms.

4.1.1 Gas Chromatography

The gas chromatograph is a heated unit that has thin silica capillary columns, which have a cross linked silicone layer. The opiate sample is injected into an inlet and heated. The sample is heated until the boiling point of the last part of the opiate sample has been exceeded by approximately 20 ËšC. This is typically between 200 – 260 ËšC. This leads to the vaporisation of the opiate sample.

The vaporised opiate sample will move to the beginning section of the silica capillaries. This is aided by an inert gas, typically helium. The temperature is somewhat reduced at the silica capillaries, typically 120ËšC. This will result in the condensation of the opiate sample. The reason for this condensation step is to ensure that all of the constituents of the opiate sample commence forward from a uniform point.

The opiate molecule will start to disintegrate as it moves along the capillary column. This disintegration is caused by varied physiochemical interactions that occur with the different constituents of the opiate molecule, during the stationary phase.

The time taken for each constituent or metabolite to move of the opiate sample to move through the capillary tube, from the point of injection is referred to as the retention time. [28]

4.1.2 Mass Spectrometry

Once the separate constituents of the opiate sample leave the capillary column, they begin to enter the mass spectrometer. The compartment between the gas chromatograph and the mass spectrometer is under high vacuum, which have quadrapoles that cover the end of the silica capillary.

Now that the sample is moving along from the GC they are met by a beam of electrons, resulting in the sample to become ionised. The quadrapoles split the different constituents of the opiate samples, in relation to their electrical charge and their molecular weight. An electrical pulse is generated as the ion detector acts on the charged opiate constituents. This is all recorded on to library computer, which generates a spectrum of the opiate constituent’s behaviour within the mass spectrometer. [29]

A Typical GC/MS A capillary tube

COMPUTER

4.2 A typical GC/MS procedure on hair samples of opiate abusers.

4.2.1 Typical GC Conditions

The type of capillary column used to quantification of the opiates is a HP 5MS, 5% phenyl methyl siloxane, with dimensions of 30m x 0.25m x 0.25µm film thickness.

The temperature of the inlet is set to 230°c.

The inert gas used was 99.999% helium, which flows at a rate of 1ml/min.

The temperature of the oven is held at 150°c for 1 minute.

The GC is then programmed to increase the temperature in the following increments. [30]

 

4.2.2 Mass spectrometry Conditions

The mass detector was set up to operate at voltage of 70eV.

The temperature at the quadrupol

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