Mohammad Yousuf Khan1, 2* Maria Qureshi3, Taha Nazir2, 4, Nisar-ur-Rahman5, M Khan6
1. Division of Pharmacy & Pharmaceutical Science, School of Applied Sciences, University of Huddersfield, Huddersfield, HD1 3DH, UK 2. Faculty of Pharmacy, University of Sargodha, Sargodha 40100 Pakistan 3. Department of Special Education, University of The Punjab, Lahore, Pakistan 4. Intellectual Consortium of Drug Discovery & Technology Development, Saskatoon SK Canada 5. Faculty of Pharmacy & Alternative Medicines, Islamia University of Bahawalpur, Pakistan 6. School of Pharmacy, University of Alberta, Alberta, Edmonton, Canada
Keywords: The aim of the present study was to explore the potential effect of the coating material on the aerodynamic particle size distribution (APSD) of formoterol from Oxis Turbohaler® using mixing inlet with Andersen Cascade Impactor (ACI) operated at flow rate of 60 Lmin-1. As the aerodynamic properties of the emitted dose from a dry powder inhaler (DPI) are usually flows dependent but have not been calibrated for low flow rates at yet. We have used novel methodology to measure these at even low flow of 28.3 Lmin-1. The Andersen Cascade Impactor (ACI) designed for 60 Lmin-1 was adapted to include a mixing inlet (MIXINLET) which allows inhalation flows through the DPI from 5 to 60 Lmin-1. The mean (SD) Mass Median Aerodynamic Diameter (MMAD) for no coating, silicone, 100% and 50% glycerin, 100% and 50% propylene glycol was 2.17 ± (0.06), 1.40 ± (1.23), 2.00 ± (0.42), 2.10 ± (0.10), 3.20 ± (0.00) and 3.17 ± (0.06) µm respectively. The geometric standard deviation (GSD) values for no coating, silicone, 100% and 50% glycerin, 100% and 50% PEG were 1.70, 0.90, 2.30, 2.53, 1.80 and 1.83 respectively. The mean ± (SD) fine particle dose (FPD) for no coating, silicone, 100% and 50% glycerin, 100% and 50% PPG was 32.31 ± (8.19), 21.69 ± (18.83), 21.13 ± (0.06), 3.86 ± (0.10) and 2.55 ± (0.05) respectively. The one way ANOVA with the application of Bonferroni’s correction was used to compare the aerodynamic droplet characteristics of the formoterol. The results indicate a significant difference between aerodynamic PSD when different coating materials were used. The MMAD was highest for PPG making it a suitable coating agent compared to other coating materials.

Coating material, Particle size distribution, ACI, Turbohaler, Formoterol.

Article Information

Identifiers and Pagination:
First Page:165
Last Page:178
Publisher Id:JAppPharm (2011 ). 3. 165-178
Article History:
Received:January 3, 2011
Accepted:March 15, 2011
Collection year:2011
First Published:April 13, 2011

In-vitro characterization of device-related parameters such as total emitted dose (TED), fine
particle dose (FPD), fine particle fraction (FPF), mass median aerodynamic diameter
(MMAD) and geometric standard deviation (GSD), is necessary while assessing the potential
clinical output of the dry powder inhalers (DPIs) or making comparison with other DPIs as
they are indicative of potential clinical performance. The aerodynamic characterisation of the
dose emitted from DPIs is, primarily, based on the degree of the de-aggregation of the
metered dose inside the inhaler during inhalation manoeuver. The de-aggregation of the
metered dose inside the inhaler occurs, predominantly, by the turbulent energy and thus,
turbulent energy is produced by the interaction of the inhalation flow and the inhaler’s
internal resistance [1]. Flow dependent dose emission has been reported to be a property of
all passive DPIs with some more prone to this phenomenon than others [2, 3 and 4]. It has
been reported that a failure to inhale deeply and forcibly from the start of the inhalation
manoeuvre results in insufficient de-aggregation of the drug particles from carrier molecules
and less deposition of drug particles into the lungs leading to no clinical efficacy [5].
Pharmacopoeia methodologies for the in-vitro dose emission characteristics from the DPIs
usually use inhalation flow corresponding to a pressure drop of 4 kPa across the inhaler using
inhalation volume of 4L [6, 7 and 8]. Studies have reported that many patients are not able to
achieve this high inhalation flow particularly required to achieve a pressure drop of 4 kPa [9,
10 and11]. Inhalation flows are generally reduced during an acute exacerbation [12, 13] thus
the emitted dose will be reduced at a time when the patient requires extra relief from
The Andersen Cascade Impactor (ACI) is considered as a method of choice to
determine the quality of the dose emitted from inhalers. The ACI has primarily been designed
to operate at an inhalation flow of 28.3 Lmin-1 with recent modifications to allow
determinations at 60 and 90 Lmin-1. As DPIs have a different resistance depending on their
design [1] then the inhalation flows required to achieve the compendial recommended
pressure difference of 4kPa will vary. When using non-standard flows the ACI, the cut-off
diameters of the stages have to be recalculated [14, 6, 7 and 8]. Identification of the dose
emission of properties of the emitted dose at low flows will be more clinically relevant than
identifying these properties using optimal conditions as recommended by the
pharmacopoeias. Patients will receive no therapeutic dose even though they have performed
the best inhalation manoeuver when de-aggregation at low flow is not sufficient to deposit
drug particles into lungs.
In order to measure the particle size distribution from a DPI at low inhalation flow, we have
adapted the ACI methodology using mixing inlet. Fig.1 shows the mixing inlet. The central
tube is surrounded by a sheath into which the supplementary air is introduced and has the
same internal dimension as the induction port. The thickness of the central tube usually tapers
towards the bottom such that it ends as a knife sharp edge. This together with the internal shallow angles ensures minimal turbulence when the two flows meet. We have adapted the
ACI designed to be operated at 60 Lmin-1 with supplementary air supplied through side arm
of the mixing inlet. The difference between these is the inhalation flow is the inhalation flow
usually drawn through the DPI. We have explored the total drug output and particle size
distribution of formoterol fumarate from an Oxis Turbohaler (Astra Zeneca, UK) using the
coated plates with silicone, 100% and 50% glycerine, 100% and 50% propylene glycol and
no coating.
Formoterol, 2 agonist, occurs as a racemic mixture, which arformoterol the R, R-enantiomer,
is the active form. It has been suggested that stereo selective metabolism and excretion may
account for the individual variation in duration of effect seen with formoterol, although the
exact mechanism remains unclear. It is potentially used in the treatment of chronic
obstructive pulmonary disease (COPD) and asthma. Inhaled formoterol is rapidly absorbed. It
is largely metabolized by glucuronidation and O-demethylation, with about 10% being
excreted in the urine as unchanged drug. The mean terminal elimination half-life after
inhalation is estimated to be 10 hours.
1. Andersen Cascade Impactor with the mixing inlet valve
The ACI designed to be used at 60 Lmin-1 was used so the stages 0 and 7 of the CI were
replaced by -1 and -0 (Copley Scientific, UK). The collection plates were sprayed with
silicone lubricant (Pro-Power Silicone Lubricant, Premier Farnell plc, UK), 100% and 50%
glycerine, 100% and 50% propylene glycol and with no coating. The 100% coating was left
to dry for 1 hour prior to analysis, whereas the 50% coating was left to dry for 30 min. The
ACI stages were assembled with coated plates and a GF/A filter (Whatman plc, UK) was
placed in the final stage of the ACI. The preseparator was filled with 10 ml of 60% methanol
(in water). Pharmacopoeias [6, 7 and 8] recommend the use of pre-separator for dry powder
inhalers (DPIs) to capture the large particle usually ? 10 µm [15]. The mixing inlet (Copley
Scientific, UK) was fixed between the induction port and the pre-separator as shown in Fig.2.
The figure illustrates how a flow of 40 Lmin-1 was drawn through the Turbohaler with the
ACI operated at 60 and 10 Lmin-1 of supplementary air provided into the mixing inlet
through its side arm. The ACI was connected to a Critical Flow Control Model TK2000
(Copley Scientific UK) to ensure sonic flow and provide the required inhalation flow and
volume. The vacuum flow was provided by HCP5 High Capacity Vacuum Pump (Copley
Scientific, UK).
2. Measurement of pressure drop (P) across the DPI
The standard pharmacopoeia method was modified using an adaptation of methodology [1] to
measure the pressure drop across the inhaler at flow rate of 60 Lmin-1. The adaptation method
is described in Fig.3. The standard mouth piece adapter was replaced by a specially made
adapter to allow an outlet to be connected to the MKS Baraton Type 223B pressure
transducer (MKS Instruments, GmbH). So the Turbohaler was encased in a specially made
chamber which was connected to the transducer. Leak test was carried out to ensure airtight
sealing of the ACI. The inhaler chamber was also connected to the MKS Type 1500 Mass-
Flow Meter (MKS Instruments, GmbH) in order to measure the mass flow through the
inhaler. The flow rate through the ACI was usually set at 60 L min-1. Thus the pressure drop
(P) across the inhaler determined was 4 kPa. 3. Determination of the aerodynamic characteristics of the emitted dose from DPI
The aerodynamic characteristics of formoterol fumarate from an Oxis Turbohaler (nominal
labelled dose of 12 µg formoterol fumarate dose and nominal emitted dose of 9 µg; Astra
Zeneca, UK) were determined using inhalation flow of 60 L min-1 with an inhalation volume
of 4 L. For each determination 10 consecutive doses were actuated into the ACI and each
dose was loaded according to the manufacturer’s recommended patient instructions. To
overcome the variability of the dose emission from the Turbohaler, 10 separate doses were
used [16]. The mean of the 10 doses, hence, limit any influence from erratic dose emission.
Three separate determinations were made for each coating.
Quantification of formoterol
All the plates and stages of the ACI, the induction port and pre-separator, after each
determination, were washed with 60 % methanol (in water). The amount of formoterol
fumarate from these washings was analysed using high performance liquid chromatography
(HPLC). The mobile phase used was acetonitrile : 5 mM disodium hydrogen orthophosphate
buffer (70:30 v/v) adjusted to pH 3 with orthophosphoric acid. The mobile phase was filtered
through a membrane filter (47 mm diameter, pore size 0.25 um) and sonicated under vacuum
for 10 min prior to use. The chromatographic studies were carried out at 25oC on a C18
Sphericlone® (250 mm × 4.6 mm × 5 µm) column (Phenomenex, UK). The mobile phase was
delivered at a flow of 1.0 ml min-1 using the injection volume of 1oo µL. UV detection was
set out at 214 nm. The calibration curves were quite linear (r2= 0.9987) for formoterol
concentrations ranging from 10 to 100 µg ml-1 (n=5). The method showed an accuracy of 99%
and intra- and inter-day precision CV of ? 1.9% and ? 1.2%. The limit of detection (LOD)
and lower limit of quantification (LLOQ) of formoterol were found to be 2.5 and 10.1 µg ml-1
4. Data analysis
The Copley Inhaler Testing Data Analysis Software (CITDAS version 2.0) was used to
determine the dose emission characteristics. The total emitted dose (TED) was the amounts
deposited in the induction port (USP Throat), the mixing inlet, the pre-separator and all the
stages of the ACI. For each determination the software confirmed that the spread of each
aerodynamic particle size distribution was unimodal and also log normal such that the
parameters can be easily calculated. TED has also been expressed as a percentage of the
nominal (labelled) dose. The amount of drug deposited in the induction port, mixing inlet and
pre-separator was calculated as emitted dose pre-impactor (EDPI).
A plot of the logarithm of the percentage less than stated size on a probability scale against
the logarithm of the effective cut-off diameter of the stages was made according to that
recommended in the Pharmacopoeias. The fine particle dose (FPD) was the amount
calculated from the cumulative percentage drug mass associated with the particles ? 5µm.
The fine particle fraction (FPF) was FPD divided by nominal (labelled) dose and expressed in
%. The mass median aerodynamic diameter (MMAD) was the size corresponding to the 50th
percentile of the cumulative mass-weighted distribution. MMAD values were calculated by
interpolating the data points closest to 50th mass percentile. As the bulk of the aerosol was
present in this region of the distribution, then the estimates of the FPD and MMAD are as
precise as possible for this method. The geometric standard deviation (GSD) was determined
as the square root of the ratio of 84.1 to 15.9 mass percentiles of the aerodynamic particle
size distribution [17].
5. Statistical analysis
Descriptive statistics are presented for the endpoints and for a comparison between these end
points, their mean difference were thus calculated. The latter were usually obtained from a
one way ANOVA that compared the six different coating conditions (No coating, silicone,
100 and 50% glycerine, 100 and 50% propylene glycol) with respect to FPF%, MMAD,
TED, FPD and GSD using SPSS V 19.0 (SPSS Inc., Chicago, IL) using the Bonferroni
correction test. From this difference the mean difference (95% confidence interval) were
obtained for each coating compared to No Coating. The table 2 shows the mean difference
(95% confidence interval) for No Coating compared to other coating materials.

1. Aerodynamic characterisation of the emitted dose
Table 1 summarises the aerodynamic characteristics of the emitted dose from formoterol
Turbohlaer. The fig.5 shows the log aerodynamic size distribution of dose emitted from Oxis
Turbohaler when the collection plates were coated with silicone, 100% and 50% glycerine,
100% and 50% propylene glycol and no coating. The data in the Table 1 summaries the effect
of coating material on the dose emission properties of the formoterol emitted from
Turbohaler. This table shows that how aerodynamic properties of the product are
significantly changed when the different coating materials are applied on the collection plates
of the ACI. The 100% propylene glycol coating ensures the increased deposition of the drug
particles on the collection plates and reduced rebounce back of the fine particles. The table 2
explains the mean difference (95% confidence interval) for No Coating compared to other
coating materials when the stages were coated with different coating materials. The data in
the table 2 also shows the probability difference revealing the comparison among the
different aerodynamic characteristics of the formoterol turbohaler. The data reveals that the
coating of collection plates with 100% propylene glycol increases the fine particle dose
(FPD) thus leading to increase the fine particle fraction (FPF) of the drug particles having a
diameter equal to or less than 5 µm. The mass median aerodynamic diameter (MMAD) is 3.2
micron ensuring the greater deposition of drug into the alveoli [15] resulting in enhanced
therapeutic effects. Fig.6 shows the TED and FPD comparison of formoterol with respect to
different coating materials. The TED for no coating, silicone, 100% glycerine, 50%
glycerine, 100% propylene glycol, 50% propylene glycol and the graph shows the TED was
very low when the collection plates were coated with 50% glycerine. The TED was 5.93 µg
when uncoated plates were used. Pharmacopoeias specially recommend the use of coating
when dry powder inhalers (DPIs) are characterised [6, 7 and 8]. The potential effect of
coating is to possibly prevent the rebounce back phenomenon of the fine drug particles when
they impact with some velocity on the surface of the collection plates. Similarly the fig.7
shows the comparison between the fine particle fraction (FPF %) and the TED (% nominal
dose). While the MMAD comparison is shown in fig.6. The smaller MMAD of formoterol is
due to the increased fraction of fine particles [5] describing that the drug particles are striking
on the surface of the collection plates with great inertia thus resulting into increased fine
particle fraction.
The smaller is the MMAD and greater the FPD and FPF, the greater will be the lung
deposition and more likely the therapeutic effect. The above figure represents the total
emitted dose (% nominal dose) that was impacted into the Andersen Cascade Impactor (ACI)
at flow rate of 50 Lmin-1 drawn through the DPI and 10 Lmin-1 was supplied through mixing
inlet. The FPF was 68.4% with TED of 9.22 µg for 50% propylene glycol coating. The
MMAD (1.4 µm) for silicone coating is too small to allow the drug deposition deep into the
lungs. The 100% propylene glycol coating thus producing the fine particles having a MMAD DISCUSSION
By choosing a suitable coating agent which would be compatible with analytical methods,
provides advantages such as prevention of bouncing back of fine particles, reducing wall loss
and represents a true measurement of particle size distribution of particles. Studies have
shown that glycerine and silicone have an equal effect to eradicate bouncing of the particles
when using the ACI [18] while, others have recommended propylene glycol as a coating
agent [19]. In this study four different types of coating agent were examined (silicone,
glycerine, propylene glycol and no coating agent) when using the ACI. Each method was
carried out ten times and in each run ten separated doses of formoterol from the Oxis
Turbuhaler® were used. The results showed that the mean (S.D.) total emitted dose (TED)
and fine particle fraction (FPF%) of formoterol emitted from Oxis Turbuhaler using
propylene glycol 100% and 50% was 9.22 (0.6) and 4.59 (0.9) and that of FPF was 68.3 %
(0.2) and 71.7 % (10.6) respectively. A decrease in the TED and FPF% was observed when
silicone was used as the coating agent where the mean (S.D.) TED 4.83 (2.1) 36.2% (1.5)
respectively. When the ACI was used without using a coating agent, the percentage of TED
(µg) and FPF% was found to be higher than silicone. The reduction in the percentage of TED
and FPF with silicone could be due to a difficulty in the extraction of the drug from the non
polar coating agent (Silicone). Although formoterol is readily water soluble and binds to
silicone, the recovery is decreased. Alternatively, non-polar solvents such as cyclohexane
being a non-toxic solvent could be used to achieve high recovery. But this solvent would
affect the C18 columns as well as the sensitivity of the HPLC system. The TED of glycerine
100% and 50% was 4.21(0.4) and 2.36 (0.1) and the FPF was that of 60.6% (7.3) and 67.8%
Unlike silicone or no coating agent, 100% and 50% glycerine, propylene glycol gave more
consistent MMAD values. Propylene glycol therefore is a suitable coating agent for the
measurement of the particle size distribution of formoterol through the ACI stages. Reduced
particle bouncing and wall loss would also be achieved by using propylene glycol and this
could be perceived as a good advantage. All types of impactors have been shown to exhibit
considerable wall losses, especially in the top few stages [20, 21]. The wall loss and particle
bouncing depends on the nature of the impaction surface, the type of coating agent, the type
of particles, particle loading on the impaction surface, the sampling conditions and the
designs of impaction substrate [22, 23]. The results highlight that wall loss was minimal. The
minimization of the effect of particle bouncing and wall loss from the impaction surfaces is a
critical factor to obtain reliable particle size distributions. Studies have shown that the coating
agents can reduce the particle bouncing and wall loss, leading to improved collection
efficiency when using the Andersen Cascade Impactor [24. Other advantages of choosing
propylene glycol as the coating agent is its lack of UV absorbance thus no interfering peaks
would be noticed with the formoterol peaks. In conclusion, optimizing the efficiency of the
ACI is important in the evaluation of the particle size distributions of the drug particles
emitted from inhaler. This study has revealed that propylene glycol is a suitable coating
Studies have revealed that for particles to stick to impaction surfaces, high-viscosity grease
coatings have been used. As particles accumulate on the surfaces, the efficiency of grease
coatings decreases rapidly with particle loading [25]. Particle build-up on impaction surfaces
may also affect the flow stream such that smaller particles get collected prematurely,
resulting in a larger MMAD [26].
Table 2 shows the mean difference (95% confidence interval) for flow rate at 60 L/min that
compares the coating agents with a non-coating agent with FPF, MMAD, emitted dose and
GSD and was determined by analysis of variance. Significant differences were observed for
the FPF% with coatings of PEG and silicone (P <0.05) which suggested a better particle size
distribution (PSD) at various stages of the ACI. There were no significant differences seen
between coatings of glycerine with no coating.
Table 1 also represents the geometric standard deviation (GSD) measure of the variability of
the particle diameters within the aerosol. An aerosol with a GSD of <1.2 is described as
monodisperse (uniform diameter distribution); an aerosol with a GSD >1.2 is described as
polydisperse (heterogeneous particle distribution). The Oxis Turbohaler had a smaller
measured particle diameter distribution (GSD) for 100% propylene glycol (1.8) compared
with other coating agents indicating particles generated from the turbohaler were more of a
uniform size.
The aerodynamic particle size distribution characteristics of formoterol Turbohaler were
enhanced using the 100% propylene glycol considered as a suitable coating material for Invitro
characterization of Formoterol Turbohaler using Andersen Cascade Impactor (ACI).
The reduction in the percentage of TED and FPF with silicone could be due to a difficulty in
the extraction of the drug from the non-polar coating agent (Silicone). Although formoterol is
readily water soluble and binds to silicone, so the recovery is decreased. While there was no
significant result was observed in PSD of formoterol employing silicone and 100 and 50%

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© 2016 The Author(s). This open access article is distributed under a Creative Commons Attribution (CC-BY) 4.0 license. You are free to: Share — copy and redistribute the material in any medium or format Adapt — remix, transform, and build upon the material for any purpose, even commercially. The licensor cannot revoke these freedoms as long as you follow the license terms. Under the following terms: Attribution — You must give appropriate credit, provide a link to the license, and indicate if changes were made. You may do so in any reasonable manner, but not in any way that suggests the licensor endorses you or your use. No additional restrictions You may not apply legal terms or technological measures that legally restrict others from doing anything the license permits
Editor in Chief
Prof. Dr. Cornelia M. Keck (Philipps-Universität Marburg)
Marburg, Germany


Welcome to the research group of Prof. Dr. Cornelia M. Keck in Marburg. Cornelia M. Keck is a pharmacist and obtained her PhD in 2006 from the Freie Universität (FU) in Berlin. In 2009 she was appointed as Adjunct Professor for Pharmaceutical and Nutritional Nanotechnology at the University Putra Malaysia (UPM) and in 2011 she obtained her Venia legendi (Habilitation) at the Freie Universität Berlin and was appointed as a Professor for Pharmacology and Pharmaceutics at the University of Applied Sciences Kaiserslautern. Since 2016 she is Professor of Pharmaceutics and Biopharmaceutics at the Philipps-Universität Marburg. Her field of research is the development and characterization of innovative nanocarriers for improved delivery of poorly soluble actives for healthcare and cosmetics. Prof. Keck is executive board member of the German Association of Nanotechnology (Deutscher Verband Nanotechnologie), Vize-chairman of the unit „Dermocosmetics“ at the German Society of Dermopharmacy, active member in many pharmaceutical societies and member of the BfR Committee for Cosmetics at the Federal Institute for Risk Assessment (BfR).

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Abbreviation: J App Pharm
doi: http://dx.doi.org/10.21065/19204159
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