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Asthma and chronic obstructive pulmonary disease are common lung diseases that can be treated systemically or locally by the administration of a corticosteroid and/ or bronchodilator to the lungs, via an inhaled route.
The inhaled route has been chosen as the most suitable route for delivering bronchodilators and corticosteroids to patients suffering from asthma and chronic obstructive pulmonary diseases (COPD). Chrystyn (2006) found that the inhaled route allows delivery of a small but therapeutic dose of drug directly to the airways, achieving a high local concentration within the lung, and minimum side effects when compared with the oral or parental route of administration.
Asthma is a chronic condition in which the airways become blocked or narrowed, causing breathing difficulty. A cough producing sticky mucus can also be symptomatic of asthma. The symptoms often appear to be caused by the body's reaction to a trigger such as an allergen (commonly house dust and animal dander) or an irritant (such as cigarette smoke or workplace chemicals). These triggers can cause the asthmatic person's lung to release chemicals that cause inflammation of the bronchial lining, leading to constriction of the bronchial wall and then a bronchial spasm. If the effect on the bronchi becomes severe enough to impede exhalation, carbon dioxide can build up in the lungs and might lead to unconsciousness or death. An obstruction can be either spontaneous or pharmacologically induced. In most countries asthma affects between 4% to 8% of the population. The main reasons for asthma are unclear, but changes in lifestyle and environmental factors such as air pollution are considered as contributing factors (Davies et al, 1997).
Asthma is an immunological disease which causes difficulty in breathing. It is a form of type 1 hypersensitivity in which the bronchioles in the lungs are narrowed by inflammation, causing a spasm of the lining airway wall. Inflammation occurs when irritated tissue swell and produce extra mucus, developing a condition known as bronchoconstriction (National Institute of Health, 1995; National Institute of Heart, 1997). The combination of changes in lifestyle and environmental factors can cause constriction or complete blockage of the airway, and can initiate symptoms of an asthma attack. Symptoms of an asthma attack can comprise wheezing, coughing, chest tightness and tininess of breath. During the normal breathing air is taken in through the nose and mouth. It goes down the windpipe, through the airway and into the air sacs. When breathing out, air is expelled from the lungs in the reverse order. During an asthma attack, the muscle around the airways tightens, making the opening in the airway smaller. The lining of the airways swells due to inflammation and mucus production, leading to blocking of the airways. Because it is more difficult to breathe out than to breathe in, more air is retained in the air sacs in the lungs with each breath.
1-1 Types of asthma
There are various different types of asthma, some of which are difficult to diagnose. Further complicating accurate diagnosis, in certain individuals, there are very specific symptoms (or patterns of symptoms) unique to any one person.
Exercise -induced asthma
Steroid-resistant asthma (severe asthma)
The term COPD completely cover a number of different disease processes, which is characterised by airflow obstruction due to a combination of damage to the airway and lung tissue. The main types of COPD include chronic bronchitis and emphysema (Helm, 2006).
Chronic obstructive pulmonary disease (COPD) is a major cause of mortality worldwide, and is the only leading cause of death predicted to increase over the coming year. In the most of clinical cases the primary cause is smoking. The death risk from COPD in patients who smoke 30 cigarettes per day is 20 times more than for a non smoker. COPD is a slower progressive disease usually following many years of smoking, although other risk factors may also be responsible.
1.2.1 Difference between asthma and COPD
Although COPD and asthma have similar symptoms such as the symptom of coughing and wheezing, they are two different conditions in terms disease beginning, rate of recurrence of symptoms and reversibility of airway obstruction.
The onset of asthma generally occurs during childhood or adolescence (National Institute of Heart, 1997). COPD most often occurs in smokers and former smokers who are in their mid-50s (Petty, 1995).
Sever of asthma -characterised by recurrent of wheezing, chest tightness, breath shortness and cough (National Institute of Heart, 1997). However, exacerbation in COPD patients are commonly caused by respiratory infections (Barnes, 1999).
Production of neutrophils in COPD patients appeared to be more than in patient who are suffering from asthma. In asthmatic patients osenophils is more when compared to patients suffering from COPD.
The aim of treatment for asthma patients to have near-normal lung function and be symptom-free (National Institute of Heart, 1997). COPD patients commonly have an experience with sym ptoms. Air flow obstruction is partially reversible in COPD sufferers.
Progression of lung function and breathlessness accelerated upward during smoking cessation whilst using of bronchodilator leading symptom relief (BTS, 2003). Despite these differences, COPD is frequently misdiagnosed, and individuals with COPD are treated instead for asthma (BTS, 2003). In piece of evidence, a survey of 75 primary care physicians publicized that even know the appropriate treatments vary, they prescribe similar medications for COPD and asthma (Kesten&Chapman, 1993).
1.2.2 Treatment of stable COPD
Abroad out line to treat and stabilise the COPD has been recommended by National Institute for clinical Excellence (NICE) as the following:
Short-acting betaâ‚‚ agonists( e.g. salbutamol, terbutaline)
Long-acting betaâ‚‚ agonists(e.g. salmeterol, formeterol)
Antimuscaranics (e.g. ipratropium bromide)
Example for that fluticasone and budesonide
1-2 Anatomy of the respiratory tract
The respiratory system consists of a conducting portion that leads gases into a respiratory portion i.e. trachea, bronchi, terminal and respiratory bronchioles, whilst the respiratory peripheral region is made up of the respiratory bronchioles and alveolar region, as shown in Figure 1 below. The airways progressively decrease in diameter, but increase in number, thus increasing total surface area as you go further down the respiratory tract. (Aulton, 2006).
The receptors for the drug used in this study i.e. Î’â‚‚ agonist, are found throughout the lung; however the drug will only induce bronchodialtion where smooth muscle is found (Al-showair et al, 2007 and Usmani et al, 2005). Predominantly smooth muscle is found in bronchioles. Therefore for efficient pulmonary drug deposition the drug should be deposited uniformly between the large and small airways. Aerosol particle size to reach these areas is required to be< 5-6Âµm. Particles less than 2Âµm in size are more likely to be deposited in the alveolar region and to enter the systemic circulation, whilst larger particles > 6Âµm will impact at the back of the throat and be rapidly cleared from the lung by mucociliary action and are subsequently swallowed (Aulton, 2007 and Taylor, 2002).
1.3 Î²â‚‚ Adrenoreceptor agonist- salbutamol
Bronchodilators are a popular class of drugs prescribed in general practice for
Respiratory conditions and include not only Î²â‚‚ agonists, but xanthines (e.g. theophylline), antimuscaranic bronchodilators (e.g. ipratropium and leukotriene), and receptor antagonists (e.g. montelukast) (BNF 56, 2008).
Salbutamol sulphate is classified as a highly selective short acting Î²â‚‚-adrenoreceptor agonist used in the treatment of asthma and other conditions associated with reversible airways obstruction, as well as in the irreversible condition of COPD (BNF 56, 2008). These groups of sympatomimetic drugs including salbutamol and terbutaline are the safest and most effective drugs for immediate relief of bronchospasm on an as required basis due to their selectivity. When high doses are administered, this specificity is lost resulting in tremors and tachycardia (Î²â‚-receptor stimulation). When salbutamol is given via inhaled route, a rapid and local effect is produced -with duration of action of around 6 hours. If given orally this effect takes 30 minutes to commence (Martindale, 2008). Oral dosage forms of salbutamol have a very good bioavailability (of 85%), but require double the time to reach peak plasma levels (Mak, 1998). So the inhaled route is preferred, especially in acute cases.
Due to the widespread use of salbutamol as the drug of first choice in step-wise cascade for asthma therapy (BTS guidelines, BNF 56, 2008), it is the chosen drug for this experiment to investigate aerosol particle behaviour at different flow rates. The chemical structure of salbutamol sulphate can be seen in Figure ---
1-4 Factors affecting deposition of inhaled particles in the lungs.
The successful deposition of a drug within the lungs is dependent on a number of factors including physicochemical properties of drug and formulation, the types of inhaler device, and the patient. Patient factors are more variable and include differences in lung anatomy, age, disease state, and inhalation technique.
1-4-1 Inhaler devices
Since the early 1930's diverse types of inhaler devices have been used to deliver bronchodilator aerosols. The foremost types of inhaler devices are pressurised metered dose inhalers (pMDIs), dry powder inhalers (DPIs), and nebulisers. The later will be mentioned briefly. Nebulizers are the oldest system and have been used in inhalation therapy since the early 20th century. Nebulizers are proper form of administration if the patient requires a high dose therapy or has a problem with using inhaler devices efficiently. Nebulizers are used for drug solutions or suspensions, which are aerosolized either by air jet or ultrasonic energy (Grossman, 1994).
The pMDI first came onto the market in the United states in 1956 (Crompton, 2004). MDIs allowed topical therapy treatment with corticosteroid and Î²-agonists to replace oral treatment in asthmatic adults (Koskela et al, 2000). The pMDIs are still the most frequently prescribed inhaler device worldwide and constitutes more than 80% of the worldwide market (O'Connor, 2004). The main types of pMDIs contain a drug which is either dissolved or suspended in liquid propellant mixture, contained within a pressurized canister fitted with a metering valve (see Figure).
Upon the actuation the valve is opened and a dose is released. The propellant droplet exits the device at high velocity (in excess of 30m/s). As the propellant evaporates, the droplet size decreases from an excess 40Âµm to that suitable for deep lung deposition (2-5Âµm), as the drug particles are aerosolized. This process can continue up to 5 seconds after actuation (Taylor, 2002). The pMDIs are cheaper, portable and have a reproducible consistency of dose. The pMDI also has the advantage of being independent of the inspiratory need to generate the medicated aerosol cloud containing the available dose for the patient. This greatly benefits patients who have low inspiratory flow due to poor lung function
(Assi et al, 2006). Although pMDIs have many problems connected with their use, they are still one of the most important respiratory delivery system. There is often poor synchronisation between actuation and inhalation especially in elderly and paediatric patients (Orehek, et al, 1976). In addition a high oropharyngeal deposition caused by the high velocity of inhaled particles which induces a local and systemic side effect (Smith, et al, 1983). There are also concerns about the adverse effect of chloeoflurocarbon (CFC) on the ozone layer and the environment, resulting in restrictions on the continued production of CFC aerosol formulations.
Dry powder inhalers have appeared to many to be an obvious replacement for the delivery of drugs via pMDIs using CFC propellants. The simplicity and the ability of new DPIs to deliver multiple doses of drugs over a long period of time, has established dry powder devices as a major competitor to the pMDIs. DPIs consist of either micronized drug particles mixed with a carrier substance (usually lactose) e.g. Accuhalers, or as pure drug e.g. Turbohalers (Feddah, 2000).
The main advantage of the DPIs is that it is a passive breath-actuated device so no co-ordination is needed between actuation and inhalation (Copley, 2007). However, the main disadvantage of the DPIs is that they are dependent on the patient's inspiratory effort to disaggregate the powder particles and generate aerosolized drug particles, of the optimum size, to reach the target sites in the lungs. Consequently the inhalation flow rate can dramatically affect the drug dose emitted from the device and the amount of dose remaining in the device. Thus, in older people, pre-school children or those with severe airway limitation; reduced inspiratory flow can result in poor drug release and low pulmonary drug deposition (Virchow, 2008).
1.4.2 Inhalation technique
Inhalation technique is an important factor affecting lung deposition and clinical response. The number and the complexity of the actions or steps undertaken to deliver the aerosol, differ from one device to another. Incorrect handling is more likely to occur with a system that requires a high number of complex actions (e.g., Nebulizers), than with less complex systems such as pMDIs and even simpler devices such as DPIs.
Studies have shown that between 50% and 90% of patients are unable to use pMDIs correctly. In one study that classified types of inhalation errors, 54% of patients had difficulty co-ordinating inhalation with actuation, 24% stopped inhalation upon release of the aerosol due to the cold Freon effect, and 12% breathed in through the nose whilst actuating the inhaler in the mouth (Crompton, 1982). DeBlaquiere found that although all patients in a sample had received initial instructions in inhaler use, only 38% confirmed proper technique when measured. Even when patients have been shown how to use their inhaler correctly, their technique tends to deteriorate over a period of time. The key component of follow up follow -up care should be a review technique (Crompton, 2004). The correct pMDIs inhaler technique, together with potential errors, is summarised in Table 1. These instructions help to diminish impaction of particles in the oropharynx and enhance the effect of sedimentation in the lower respiratory tract.
Errors in technique
1)Remove the mouthpiece cap
Failure to remove cap
2) Shake inhaler(suspension only)
Inhaler not shaken
3)Prime inhaler if new or not used for
More than one week
Inhaler upside down
4)Holder inhaler upright
5)Breath out as far as comfortable to
Firing device before start of inhalation
6) Place the mouth piece between lips and
Close lips around the mouthpiece
Stopping Inhalation rate is too fast inhalation as device is fired
7) Begin to breath in slowly and fire Immediately
No/short breath hold
8) Continue to inhale slowly and deeply
Over a period of 5-10 seconds.
9) Remove inhaler from mouth and hold Breath for 10s
10) Breath out slowly
Difficulties in the manner patients use MDIs have been a cause in the development of other forms of inhaler. Eliminating the need for co-ordination between actuation and inhalation, by developing breath-actuated pMDIs, has been shown to improve lung deposition and reduce oropharyngeal deposition in patients who have co-ordination problems with standard pMDIs (Newman et al, 1991). However, the inhalation must be strong enough to generate the firing mechanism (approximately 20-30 L/min), this means the patient has to have a higher inspiratory flow rate to gain benefit from the inhaler, which may not possible in an acute attack, dependent on the severity of the disease.
Spacer devices were first designed for use in association with the pMDIs in the late 1970,s (Iula etal, 1997). Their use has also offered a solution to pMDIs problems by eliminating the need for co-ordination between inhalation and actuation and reducing the primary droplet size of aerosol cloud. This happens because extra time is provided, allowing propellant, found within the pMDIs to evaporate before impacting on the oropharyngeal and thus reducing the particle droplet size (Feddah etal, 2000). Oropharyngeal deposition with actuator alone range from 30 to 70%, compared with 5-10% with spacer devices (Kims, etal, 1987, Ahrens R, etal, 1995). Large particles are more likely to impact on the side of spacer device by reducing the velocity of particles passing through spacer chamber and so deposition of drug particles is less likely to occur at the back of throat and also is likely to cause irritation. (Feddah, 2000).pulmonary drug deposition can be affected by many properties of spacer devices such as size, shape and volume of spacer, electrostatic effect, presence of valves and dead space. Electrostatic charges can reduce the drug output from the plastic spacer. This is prevented by washing the spacer out with a detergent and allowing air to dry, however no studies have been carried out to investigate if this may interface with stability of drug or reproducibility of the dose, another option is substitution for a metal spacer (Kwok etal, 2006). An in-vivo and in-vitro study has shown that both metal Nebuchamber Â® and volumatic spacer Â® are interchangeable, both achieving similar pulmonary deposition ( Terzone etal, 1999).
One of the aims of spacer devices is the removal of large particles; however there is a loss of aerosol particles. The most important causes of aerosol loss from pMDIs delivered into spacer are illustrated in fig. The large particle impact on the spacer wall due to the inertia in the jet of particles from pMDI. Particles sediment due to reducing velocity of the aerosol. In addition, particles can be adsorbed to the spacer wall if this carries electrostatic charges. To rise above this problem the inner surface of spacer has been treated by using anti static paint, ionic detergent, which is more effective than non-ionic detergent and Benzalkonium which play an essential role in neutralising the electrostatic charge of plastic spacers.
1.4.4 Particle size
The involvement of the different mechanism for lung deposition is mainly determined by particle size. The aerosols particle size from 2-6 Âµm has been considered as ideal size range for deposition throughout the airways (Hickey 1992). The aerodynamic particle size distribution (APSD) of an aerosol is defined by three parameters, the mass median aerodynamic diameter (MMAD), the fine particle dose (FPD, dose<5 Âµm in diameter) and geometric standard deviation (GSD). The MMAD represents the aerodynamic diameter of the particles and takes into account not just physical diameter but their density and velocity within air flow. The FPD represents the amount of each dose that contains particles in respirable range i.e. <5 Âµm in diameter. The GSD is a measure of the spread of the distribution. Aerosols considered mono dispersed if the GSD IS < 1.2. In study published by Usmani etal (2003) in which radiolabelled monodisperse salbutamol of 1.5, 3 and 6Âµm were given to twelve mildly asthmatic patients, the grater total lung deposition was achieved by smaller particle1.5Âµm (56%), whilst those 3Âµm and 6Âµm achieved 50% and 46% respectively. Highest peripheral lung deposition was presented from the smaller particle sizes.
1.4.5 Inhalation flow rates
Respiratory disease pathology can diminish the respiratory effort a patient can achieve and this study is trying to determine if salbutamol has been designed to take this into account. Salbutamol is a repeatedly prescribed medication for pulmonary conditions, but what this study set out to investigate if this inhaler is appropriate to administer to all patients regardless of the optimal inhalation flow rate each individual can achieve. The majority of patients using pMDIs inhaling too fast, despite continued patients counselling and further support with patient information leaflet (Al-showair etal, 2007). The recommended technique for pM DIs involves a slow and deep inhalation with a flow rate <90L/min. Only one in-vivo study has investigated the effect of a low flow rate of 10L/min with salbutamol pMDIs, it concluded that a greater bioavailability of the pMDIs associated with this low flow rate. Due to the limitation found in this study, further investigation is still required. (Tomlinson etal, 2005, Hindle et al, 1993). A literature search have found that no in-vitro studies have investigated the effect lower inhalation rate (i.e. 30L/min) have on the particle size distribution of an emitted dose form a salbutamol pMDIs. With the inter-patient variability it is important to investigate the in-vitro dosing characteristics of a range inhalation flow rate including those lower than 30L/min since any change in particle size distribution may indicate changes in the therapeutic response. This is could be consider as significant in old children <6 year old who may have lower than average inspiratory flow rates (Newhouse et al, 1999).
This research project outline the differences of the in-vitro characteristic of the emitted dose from salbutamol pressurised metered dose inhaler attached to a small volume Aerochamber plusÂ® spacer device at the following inhalation flow rates 10, 20, 28.3 and 60L/min. These four flow rates have been chosen to compare the difference between slow and fast inhalation rates. The slow flow rates 10L/min have been investigated before and may show significant difference in terms of therapeutic benefit. It would be interesting to look at different low flow rates e.g. both 10 and 20L/min to see if there is too low flow rate that would increase bioavailability of the pMDIs.
The aim of this project to investigate the effect of different flow rates 10L/min, 20L/min, 28.3L/min and 60L/min on particle size characteristics from salbutamol pMDIs using spacer device and without spacer. Also, to identify the optimum spacer for different categories of asthma.
Measure the fine particle dose (FPD), as determined by Anderson Cascade Impaction using mixing inlet, emitted from a dose from salbutamol pMDIs with an attached Aerochamber plus Â® spacer device at different flow rates (i.e. four rate mentioned above) with an inhaled volume of 4L.
Measure the mass median aerodynamic diameter (MMAD) of the particles, as determined by Anderson Cascade Impaction using a mixing inlet, of the emitted dose from a salbutamol pMDIs with an attached Aerochamber plus Â® spacer device at different flow rates (i.e. the four rate that mentioned above) with an inhaled volume of 4L.
Measure the geometric standard deviation (GSD) of the particles, as determined by Anderson Cascade Impaction using a mixing inlet, emitted from a dose from salbutamol pMDIs with an attached Aerochamber plus Â® spacer device at different flow rates (i.e. the four rates mentioned above) with an inhaled volume of 4L.
Measure the FPD, MMAD and GSD of the particles as determined by Anderson Cascade Impaction using a mixing inlet of the emitted dose from salbutamol pMDIs without attached spacer device at different flow rates (10, 20, 28.3 and 60L/min) and an inhaled volume 4L.
Compare the FPD, and MMAD of the particles emitted from a single dose of salbutamol pMDIs, with an attached small volume spacer and without small volume space at the different flow rates (10, 20, 28.3 and 60L/min) and an inhaled volume of 4L.
HPLC was used for quantification of emitted dose from salbutamol pMDIs.
The salbutamol sulphate standard and Bamethane internal standard were ordered from Sigma-Aldrich Ltd. The silicon spray was ordered from Dow Corning Ltd. The acetonitrile and methanol HPLC grade were ordered from Fisher Scientific and potassium dihydrogen phosphate was orderd BDH. The slabutamol was used a Airomer Â® CFC-free inhaler delivering 100Âµg salbutamol sulphate/ actuation. The spacer was used an Aerochamber plusÂ® (Trudell Medical International?) which has a volume of 145ml.
3.2 Operational procedure of ACI
Before running Anderson Cascade Impactor, the 8 plates were washed with methanol and dried at room temperature. These plated were sprayed with fine uniform coating of the solvent silicon in a well ventilated fume cupboard and left to dry for 35minute. The temperature and humidity were measured. The main purpose for using the silicon is to prevent drug particles bouncing off the plate and re-entering the airflow stream. The ACI was assembled and a single 8.1cm glass microfiber filter paper (whatman) placed below the final stage to capture ay fine particles that would otherwise escape from the device. The mixing inlet and USP induction port were attached. Parafilm was used to prevent any leakage between the ACI inlet one, mixing inlet and induction port. The outlet of the ACI was attached to the vacuum pump via a critical flow controller (model TPK, Copley) which has been used to control the flow rate and duration of flow through the cascade impactor.
Before each determination, the vacuum pump was switched on the flow rate through the impactor fixed to 60L/min during all experiments using a calibrated flow meter (Copley DFM 2000) attached to the induction port. The pressure difference cross the critical flow controller were checked to ensure the airflow through the impactor was stabilised and critical (sonic) flow (P3/P2 â‰¤0.5) was achieved through the flow control valve. This is ensured that flow rate through impactor and inhaler was unaffected by minor vacuum pump fluctuation. The flow rate through impactor was set at 60L/min using the critical flow controller and then compressed air was introduced into the side port of mixing inlet until the flow rate through the mouthpiece was reduce to the desired level 10, 20 and 28.3L/min. The mixing inlet was in place for all runs but when the run for 60L/min were carried out the mixing inlet valve was sealed off using parafilm laboratory film to prevent leakage.
The spacer was then attached to the induction port by using a moulded mouthpiece adaptor. Before attached the inhaler to the spacer, the pMDIs was shaken vigorously and primed by discharging two shots to waste. This is recommended in accordance with the US pharmacopoeia and the manufacturer's patient information leaflet.
The timer on the critical flow rate controller was set so that the duration of flow was adjusted to achieve an inhaled volume of 4L which represent a healthy human lung capacity for an average male weighing 70kg (see figure below for calculation). At this stage the outer valve of mixing inlet was opened and flow rate could be reduced at the desire tested flow rate by adjusting the compressed air cylinder without changing the calibration of the impactor.
Tested flow rate (L/min)
Duration timer control is set to (seconds)
Figure : Each tested flow rate is set to an individualised run time to achieve the standard
4L/min inhaled volume.
In order to obtain the desired time to allow the inhaler actuation to pass through the apparatus, it was simply divided the desired flow rate in L/min by 60 seconds to attain a value per second. Then in order to achieve equivalent volume of 4L/min, that is representative of a healthy human lung, we divided 4L/min by the previous answer. E.g. to calculate a test flow time for the 60L/min, we use 4/ 60/60 and yield a value of 4 seconds, thus the test is allowed to run for a total 4 seconds during actuation of the inhaler device. Once the timed duration of flow had elapsed, the inhaler was removed after 5 seconds, and shaken for 5 seconds in the preparation for the next actuation. This was repeated until a total 5 doses for each flow rate were actuated. A schematic diagram of the apparatus is shown in figure on the next page. Each actuation delivered salbutamol sulphate equivalent to 100Âµg salbutamol per dose into the impactor. The inhaler was removed from the mouthpiece when the solenoid valve had close and the pump had been switched off. The apparatus was then dismantled carefully to avoid nay loss of drug. The active ingredient was washed from the inner wall and collection plates of each stages into an appropriate volume of washing solution (8mg/L of Bamethane)(Table----).
Volume of washing(ml)
PMDI with spacer
The salbutamol was extracted from the filter into the washing solution. Furthermore to ensure complete extraction the filter was sonicated for 5 minute in the washing solution. Also, the washing solution from the filter was further filterd through a 0.45 Âµm filter in order to remove any undesired particles which might block the HPLC system particles which might Each tested flow rate was repeated three times to increase the reliability of the results. The determination was carried out at standard temperature and humidity. Following each determination the collection plate were cleaned with methanol to remove the silicon coating, rinsed with distilled water before being left to air dry. The spacer, induction port and mixing inlet were rinsed with distilled water and left to air dry.
The same procedure was used without spacer and inhaler was attached immediately to induction port. Variables introduced by the operator, apparatus or environment were kept at minimum.
3.3 Preparation of samples for HPLC Analysis.
The impactor was then dissembled and drug recovered from each stage by rinsing the collection plates , final filter, induction port, mixing inlet, mouthpiece and spacer in appropriate volume of a suitable solvent containing an internal standard (Bamethane sulphate8mg/L in distilled water) in preparation for HPLC assay. Bamethane was chosen as our internal standard because it has similar isomer characteristic to salbutamol and is also a weak Î²2 agonist in clinical practice. Due to sensitivity of the HPLC, the rinsing from the filter was also filtered again through a 0.22 Âµm micro filter. (MillexÂ® GP). The stages themselves were not rinsed because assumed that inter-stage wall losses were less than 5% and so only collection plates were rinsed as per pharmacopoeial methods (USP, 2005).
The rising from each stage were assayed, and the amount of salbutamol were quantified, using Performance Liquid Chromatography (HPLC) analysis by isocratic elution i.e. the composition of mobile phase remains constant through separation.
3.4 Chromatographic condition for salbutamol
Reverse phase HPLC was used to determine the salbutamol sulphate concentration. The HPLC conditions are outlined below:
HPLC System: Heweltt Packard series 1050 modil
Column: Phenomenex Sphereclone ODS (2) 5Âµm (250 * 4.6mm)
Mobile phase: 25% Acetonitrile: 75% KHâ‚‚POâ‚„ (5mM, pH 3)
Flow rate: 1ml/min
Injection volume: 50ÂµL (2 injections per vial)
Detector: UV light at wavelength 202nm
Integrator: PRIME software
The HPLC was operated at room temperature. Each sample was run for 7.25 minutes is to allow retention times of both salbutamol (3 minutes) and Bamethane (4-5 minutes). Each sample was injected twice to make sure the results more likely to be consistent and precise. I have used a HPLC method designed by (Silkstone, 1999). Assi et al 2005 proved this particular HPLC method to give a high analytical performance, taking into account sensitivity, linearity, accuracy, precision and solution stability. Quantification was by divided Salbutamol peak area ratio with Bamethan peak area ratio and transferred to an excel spreadsheet.
3.5 Preparation of Salbutamol standard
10mg of salbutamol sulphate (sigma) was weighted out on an appropriately calibrated balance. To yield 100mg/1000ml concentration, it was made up to 100ml with distilled water. From this stock solution, the sub-master solution was prepared using a 10 fold dilution i.e. 10ml of master solution was made up to 100ml volume with Bamethan 8mg/L sigma to give a 10mg/1000ml concentration
Eight serials were measured from the sub-master solution and also made up to 100ml volume with Bamethane 8mg/L
This range of concentrations i.e. 50-2000mcg/L was validated and linear calibration curve was obtained.
3.6 Preparation of mobile phase
KHâ‚‚POâ‚„ (Pottasium dihydrogen orthophosphate, sigma) 6.8045g was weighted out using calibrated balance (sensitivity 0.1mcg-25mg) and dissolved in 100ml distilled water. 10ml of this solution was taken and diluted down to 1000ml with distilled water to yield a 5mmolar solution. 750 of buffer solution was added to 250ml of acetonitrile to give 1 L of mobile phase solution. This buffer then was filtered using vacuum and degassed ultrasonically.
PH meter was used to measure the PH of mobile phase solution and two standard solution of pH 4 and pH 7 was used as comparison. Concentrated phosphoric acid was added to lower the pH to 3 to obtain correct absorbance and retention time with HPLC.
3.7 Data Analysis
The salbutamol sulphate concentration in each sample was quantified using a standard calibration curve in the range of 50Âµg to 2000Âµg/L. With the knowledge of the volume used to rinse each sample, this concentration was converted into mass quantity of salbutamol sulphate recovered on each stage of the impactor. The HPLC chromatogram data was transposed into Copley C.I.T.D.A.S (Copley Inhaler Testing Data Analysis Software) which calculated the particle size distribution for each stage, FPD, %FPF, MMAD and GSD for all flow rates.
3.8 Reliability of method
The apparatus was calibrated before each run using the flow meter to an accuracy of+/-5%. A constant sonic flow rate of 4Kpa was checked by ensuring the P3:P2 ratio <0.5 and the timer control was set for each appropriate flow rate (60sec/ flow rate* 4L) to achieve an inhaled volume of 4L through ACI. The temperature and humidity were monitored to make sure there was no change in conditions during running the experiment.
In attempt to eliminate any discrepancies in the data, two injections were taken from each sample during run the HPLC. Bank samples were put in between each set of sample to make sure the machine was working properly. The calibration curve was based on sample concentrations discovered from previous work so that accurate concentrations could be found from the samples. The manufacture's recommendations were followed during running the experiments. Each flow rate was carried out three times to make the results more reliable.
3.9 Health and Safety
Good laboratory practice principles were adhered whilst conducting the experiments. Lab coat was worn and gloves were used when handling chemical. All chemicals were stored correctly and the attention was paid toward the fume cupboard when spraying silicone. A COSHH assessment form was submitted containing all chemical used. Clean and tidy working environment were regularly maintained.
The standard calibration curve used to quantify the salbutamol sulphate concentration is shown in figure 4
Figure4 Standard calibration curve for salbutamol sulphate with rang 50mcg/L to2000mcg/L
Figure 5: Graph plotted from results gained from Copley software to discover the mean amount of salbutamol sulphate collected on each stage of cascade using pMDI with spacer at different flow rates.
Figure 5 Percentage (Nominal dose) of salbutamol MDIs deposited on each stage using pMDI with spacre at diffrent flow rates.
The histogram in the figure 5 display that the main area for salbutamol largest deposition of drug was on stage 3, stage 4 followed by stage 2. The stage 3 collected the highset amount in particles size range most likely to be deposited in the lung (i.e between 2-5Âµm). The stages with least amount of drug deposited on it were stage -1, stage -0, stage 5 and filter. The histogram also show the greateset deposition of metered dose as deposited in spacer at the flow rate 10L/min
Figure 6 Graph plotted from the data that found by inputing raw data from chromatograms into Copley software to discover the mean amount of salbutamol sulphate collected on each stage of cascade using pMDI alone (i.e with out spacer) for all three runs at diffrenet flow rates 10, 20, 28.3 and 60L/min
Figure6 Percentage (nominal dose) of salbutamol MDI deposited on each stages using pMDI alone at different flow rate
The histogram in figure 6 show that the main area for salbutamol sulphate deposition was in induction port and at 60L/min was fewer amounts when compared with other flow rates, followed by MP (mouth piece) but at 60L/min was less deposition of drug when compared with another flow rates. For the amount of drug collected on each stages, the stage 3 was highest followed by stage 4 and stage 2. The stage with least amount of drug deposited on it was stage -1, -0 followed by stage 6 and filter.
Figure 7 Histogram plotted from data by inputing data into copley software to discover % of FPD collected from salbutamol using pMDI with spacer and alone at Diffrent flow rates
Figure 7 comparison of FPD % of (Nominal dose) using PMDI alone and with spacer at different flow rate
Chart 7 illustrates the amount of salbutamol delivered as a fine particle dose (FPD, Âµg) quantity as % of nominal dose of salbutamol particles collected from stage 1 until filter. This chart shows both of FPD from spacer and MDI alone increasing gradually with increasing flow rate. The lowest FPD is seen at 10L/min when using MDI alone. At 60L/min, both pMDI alone and with spacer, delivered the most salbutamol as fine particle dose. The FPD emission of salbutamol from Aerochamber plus is higher than MDI alone at varying flow rate. I.e. the effect of spacer on FPD emission is noticeable.
Figure 8: Histogram plotted from data that found by inputting data from chromatograms into Copley software to exhibit % of FPF salbutamol using spacer and MDI alone at varying flow rates.
comparison of %FPF of emitted dose from pMDI alone and with spacer at diffrent flow rates.
The histogram in figure 8 show the % of FPF is higher in spacer than MDI alone except at 60L/min.
Figure 8: Histogram showing delivered dose of salbutmaol pMDI (%) of nominal dose with using Aerochamber plus spacer at diffrent flow rates.
Delivered dose (%) of nominal dose for pMDI with spacer at different flow rate
The chart in figure 9 present the highest delivered dose % of nominal salbutamol is 60L/min. 10L/min does not enough to deliver right dose but 28.3L/min and above are enough to deliver exact dose.
Figure 10: Histogram showing spacer deposition from a single actuation of salbutamol PMDI, at varying flow rates.
Dose in spacer (%) of nominal dose at different flow rates
The histogram in figure 10 display the largest amount of salbutamol pMDI (%) of nominal dose deposit in spacer at 10 L/min and this amount decreasing gradually as flow rate escalating.
Figure 11 Histogram gained by input data obtaind from chromatograms into Cpley sofware to show % of nominal dose of salbutatmol deposited in throat using spacer and pMDI alone.
Comparison of throat deposition (%) of nominal from pMDI alone and with spacer at different flow rates
The results displayed in figure 11 show that very little deposition of drug on throat when using Aerochamber plus spacer at different flow rate that had been used (mostly the same). However there was a high deposition of drug when used pMDI alone at different flow rates, highest deposition on throat appeared at 28.3L/min followed by 10L/min. The smallest deposition was shown at high flow rate 60L/min.
This collected work was carried out to look into the effect of changing flow rate on the behaviour and particle size distribution of salbutamol sulphate pMDI inhaler using spacer and without spacer as comparative. The results of this experiment would be clinically considerable when related to the patient using inhaler in practice, also would help to discriminate if there was any was any gain or disadvantage at particular flow rate. The change in flow rate might affect on FPD, this could cause an increase or decrease in the efficiency of drug and its local effect on bronchial smooth muscle on the flow rate. The result gained from the experiment would give suggestion of how successful this brand of pMDI is at low (>28.3L/min) and high (>60L/min), or if there were any variation at all. Results connected with low flow rate were our focus, because patients with inspiratory problem found a difficulty to attain a high rate of inspiration. In asthmatic patient, the mean peak inspiratory flow rate (PIFR) was found to be about 60L/min. However, during an acute attach decreased by 50% and this likely time for the use of salbutamol inhaler (Brown et al, 1995).
All inhaler suppliers have recommended the slow and deep inhalation when using their devices, although an exact flow rate not specified. The results found in this study have shown should not be the case. The flow rate is presented to give the best overall distribution in optimal stage around 28.3L/min.
The results showed a lower FPD in flow rate 10L/min when compared with flow rates 20L/min, 28.3L/min and 60L/min in both when using spacer and pMDI alone. Also, it was observed that the FPD increased gradually with an increasing flow rate. The results suggest there was a greater lung deposition in the respirable range (>5Âµm) seen in the higher flow rate when compared with flow rate 10L/min. This reflected in the graphs when looking at the amount of drug deposited in the optimal area stages 2, 3 and 4 of the ACI using Aerochamber plus spacer and pMDI alone. These stages resemble trachea and primary bronchi (3.3-4.7Âµm), secondary bronchi (2.1-3.3) and the terminal bronchi (1.1-2.1). The smallest amount deposited on theses stages was at 10L/min on both spacer and pMDI alone. Flow rate 20 and 28L/min have extremely the similar result w for theses three main stages when using spacer and pMDI alone. Both with spacer and pMDI alone, the amount of drug deposited on stage 3 of cascade increase as flow rate increases. It seems reasonable that the particles with greater size would have more momentum to be carried further down the cascade at higher flow rate. It would be usual that, with this increased momentum there should be an increased amount of drug deposited in throat piece (induction port). It is a thought that smooth surface of the metal throat piece causing particles to bounce off and re-enter the airstream. In future experiment it might be importance taking into account coating the induction port piece to see if there is any difference in results.