Submission: March 23, 2021; Published: April 09, 2021
*Corresponding author: Shrinivas Bhope, Sava Healthcare Limited, Research Centre, Pune, India
How to cite this article:Neeraj Jadhav, Priyanka Gondhale, Sachin Nage, Bhaskar Musmade, Shrikant Kulkarni, et al. Quantitative Estimation of Avicel
Polymer and its Impact on Spray Pattern, Plume Geometry and Droplet Size in Nasal Spray Products. Glob J Pharmaceu Sci. 2020; 8(2): 555733.
Throughout the world, the regulatory agencies like US FDA, MHRA, Health Canada recommend that during the development of generic products like Nasal sprays, Ophthalmic solutions, the inactive ingredients in the generic product formulation must be qualitatively (Q1) and quantitatively (Q2) similar to the reference listed drug product. This requires a careful deformulation/reverse engineering study of the reference listed drug product or innovator product for all the listed inactive ingredients. Avicel is one of the most important ingredients primarily used in various Nasal spray and ophthalmic suspension products as a suspending agent. It is a mixture of microcrystalline cellulose and carboxy methyl cellulose. The aim of this study is to develop a simple yet robust analytical method for the accurate quantitative estimation of this polymer for generic product development to regulatory markets. We have not found any reported method for the accurate quantitation of this polymer from any of the pharmaceutical dosage forms. Hence, this article will certainly accelerate the generic development of different pharmaceutical dosage forms for regulatory markets.
Due to its non chromophoric nature, Avicel polymer is derivatized to a readily quantifiable compound by using diphenylamine reagent. The complex is measured at wavelength 635 nm without any interference. The polymer can be successfully quantified at concentration of 0.26 μg/ml and above from various pharmaceutical products such as tablets, nasal spray suspensions, ophthalmic products etc. This paper gives a simple yet accurate and precise method for the quantitative estimation of Avicel by derivatized spectrophotometry from various pharmaceutical products.
Abbreviations: US: United State; BP: British Pharmacopeia; FDA: Food and Drug Administration; ANDA: Abbreviated New; Drug Application; RLD: Reference Listed Drug; ICH: International Conference on Harmonization; MCC: Microcrystalline Cellulose; CMC: Sodium Carboxy Methyl Cellulose; NIR: Near Infra-Red Spectroscopy; LOD: Limit of Detection; OQ: Limit of quantitation; SD: Standard Deviation; RSD: Relative Standard Deviation; FD: Forced Degradation; H: Hour
Excipients are inactive ingredients of any finished pharmaceutical products. They provide stability to the active pharmaceutical ingredients (API). The different excipients include diluents or fillers, binders, disintegrants, lubricants, colouring agents and preservatives. Sometimes these excipients help in improving the biopharmaceutical profile, appearance and patient acceptability of finished pharmaceutical products. The use of right quality, type of excipient and accurate quantity is most important during the development of any medicinal product.
The International guidelines like ICH, FDA gives more stress on the quality of such excipients. One of the most common excipient
in oral solid dosage forms is Micro crystalline cellulose (MCC). It was discovered by Battista and Smith in 1955. It was later commercialized under the brand name Avicel®
Avicel (Figure 1) is used as a binder/diluent, anti-adherent and as disintegrant in various pharmaceutical products. It is a mixture of MCC and sodium carboxy methyl cellulose (CMC). MCC is partially depolymerized cellulose and is composed of crystalline and amorphous domains . MCC is prepared by the
acid hydrolysis of cellulose . The degree of crystallinity in MCC
greatly affects its compactibility, flowability and the stability of
the medicinal product . During the manufacturing of MCC both
softwoods and hardwoods are used as a possible source . The
moisture content, particle shape and particle size distribution
varies among different grades of MCC samples. This makes a
significant difference in the stability of pharmaceutical products.
The low cost MCC grades are manufactured from various
agricultural residues , sugarcane peel, bagasse [6,7], banana
pseudo stem , groundnut shell , from cotton , corn husk
Avicel® PH 101 (CAS No.9004-34-6) is commercial brand of
MCC that is obtained from costly hard wood. Various grades of
Avicel eg. RC-591, CL-611, RC 591F are currently being used in
the pharmaceutical industry as excipients. The different grades
of Avicel mostly differ in the percent composition of MCC and
sodium CMC having different viscosity and pH.
There are stringent regulatory standards issued for nasal
dosage forms in USA  and in Europe  which recommends
that the inactive ingredients in the test product formulation be
qualitatively (Q1) the same and quantitatively (Q2) essentially
the same to the reference listed drug product. Hence, estimation
methods for excipients appear crucial since any variation in
excipients quantity would have a potential impact on the quality
and stability of the developed pharmaceutical formulation .
For the pure excipients, the method used for the quantitative
estimation by both European Pharmacopoeia and US
Pharmacopoeia for Avicel [15,16] is by using the potentiometric
titration method. This method cannot be used for the finished
pharmaceutical products because of the interference from other
excipients. Size exclusion chromatography with refractive index
detector are used, however, because of the polymeric nature of the
compound, the peak observed is not a sharp gaussian shape peak,
because of which accurate quantitation is not possible. In one of
the reported methods , the total cellulose content (MCC and
CMC) of liquid formulations like nasal spray products is calculated
by gravimetric method by determining the water content by
evaporating the liquid portion and weighing the total solid mass
obtained. The cellulose content is then determined by subtracting
the contents of all other ingredients (actives and excipients)
from the total solid. This method lacks both the accuracy and
reproducibility since it is an indirect method of estimation.
Since excipients such as MCC is crucial for any pharmaceutical
drug stability as discussed above, having its extract concentration
estimated is equally critical. From our extensive literature
search, we found merely two papers that discuss about
estimation methods for MCC. These include Near Infra-Red
(NIR) chemometric method  and an estimation method that
involves dialysis, cellulase hydrolysis, and a reducing sugar assay
. Since the NIR method lacks sensitivity and requires special
instrumentation. The hydrolysis method described by Zhang et al.
is laborious and not sensitive enough, we explored the possibility
of looking into developing estimation method of MCC that will be
sensitive, quantitative and specific too. In the present research
paper, the methodology for the quantitation of Avicel from nasal
spray suspension product is explained in detail.
AR grade Acetic Acid, Diphenylamine, Hydrochloric Acid,
Ortho-phosphoric Acid, Hydrochloric acid, Sodium hydroxide and
Hydrogen peroxide were purchased from Merck Limited, Mumbai,
India. Avicel pH RC 591 was purchased from FMC Corporation,
Philadelphia, PA 19103.
3.75 gm of diphenylamine was weighed accurately and
transferred into 250 ml volumetric flask. 150 ml acetic acid was
added and sonicated to dissolve it completely. Further 90ml of hydrochloric acid was added and mixed well by sonication. The
reagent needs to be freshly prepared before use.
About 20 mg of Avicel pH RC 591 working standard was
weighed accurately in 250 ml volumetric flask, about 175 ml
ortho-phosphoric acid was added, the mixture was heated on
water bath at 90°C till it is completely dissolved. The flask was
then removed and kept for cooling at room temperature. The
volume was made up to the mark with water, mixed well by using
the sonicator and cooled at room temperature. Further, 1 ml of
standard stock solution was diluted to 25 ml in a volumetric flask
and the volume was made up to the mark with diphenylamine
reagent, mixed well and transferred about 15 ml solution into the
test tube. Heat the test tube filled with solution in a liquid paraffin
oil bath set at 105°C ± 2°C for 90 min. The test tube is then cooled
to room temperature in ice bath for 10 min.
1 ml of 70% ortho-phosphoric acid was transferred into a 25
ml volumetric flask and diluted to volume with the diphenylamine
reagent. 15 ml solution is further transferred into the test tube.
The test tube with the solution was heated in a liquid paraffin oil
bath set at 105°C ± 2°C for 90 min. The test tube is then cooled to
room temperature in ice bath for 10 min.
The Nasal spray suspension equivalent to 20 mg of Avicel pH
RC 591 was accurately weighed and transferred into a 250 ml
volumetric flask. About 175 ml ortho-phosphoric acid was added
and the flask was heated in water bath at 90°C till it dissolves
completely. The flask was then cooled at room temperature and
volume was made up to the mark with water, mixed well and
allowed to cool at room temperature.
The placebo solution was prepared on the same line to sample
solution except the addition of Avicel. The placebo solution was
accurately weighed and transferred into a 250 ml volumetric flask.
About 175 ml ortho -phosphoric acid was added and the flask was
heated in water bath at 90°C. The flask was then cooled at room
temperature and volume was made up to the mark with water,
mixed well and allowed to cool at room temperature.
The samples of excipients like Avicel, Microcrystalline
cellulose, Sodium CMC and pharmaceutical dosage forms like
Ophthalmic suspension and Tablet are prepared as per the
procedure mentioned under sample preparation for nasal spray
suspension for the determination of Avicel content.
The optimization of the method was done by varying
the critical method parameters like Avicel concentration,
temperature, reagent concentration, heating time etc. Avicel
being insoluble in water didn’t give reproducible results. It may
be because of the non-uniformity of the sample during the second
dilution. 70% orthophosporic acid was used as a diluent to
solubilize the polymer instead of water as a diluent. This resulted
into a consistent and reproducible absorbance at 635 nm. The
wavelength maximum was selected after scanning the solution
in the entire UV visible region. Avicel does not have absorbance
in the UV region because of the non chromophoric molecular
structure. We had derivatized the molecule by reacting with
diphenyl amine reagent. The reaction gave a dark blue coloured
complex resulting into hyperchromic shift showing wavelength
maxima at 635 nm. The optimal detector response was obtained
when the reaction was carried out by heating at 90oC for 10 min.
The derivatization reaction takes place between Avicel polymer
and diphenyl amine in acidic medium (70% orthophosporic acid)
at the elevated temperature of 90oC.
The described method has been successfully validated for
parameters like specificity (selectivity and forced degradation
study), LOD (limit of detection) and LOQ (limit of quantification)
linearity, accuracy, precision, solution stability and robustness
The Specificity of the method was proved by scanning
and measuring the absorbance of diluent, Placebo solution,
Standard solution and Sample solution at 635nm for evaluating
any interference of diluent and placebo solution at the selected
wavelength. During the specificity no interference observed from
the diluent and placebo solution at detection wavelength 635nm.
Based on these observations the method is found to be selective
for Avicel at the selected wavelength.
FD study was performed to prove the specificity of the method
and to evaluate the stability indicating nature of the method.
Diluent, sample and placebo was exposed under relevant stress
conditions viz. heat, light, temperature/humidity, acid, base
and peroxide (Table 1) and the exposed samples were analysed
as per the method. The absolute % degradation of sample was
determined (Table 2). During the FD study, it was observed that,
treated diluent and placebo by various FD conditions did not
show any interference on quantification of Avicel at the detection
The LOD is the point at which the signal from the analyte
is equal to three times the noise in the measurement. The LOQ
is the lowest concentration of analyte that can be determined
with acceptable precision in sample matrices. A series of low
concentrations from 0.034 μg/ml to 1.686 μg/ml for Avicel were
prepared based on standard response and the absorbance was
recorded in triplicate. The calibration line curve was prepared for
absorbance vs concentration. From the calibration curve slope,
intercept and correlation coefficient along with the STEYX was
determined for the calculation of LOD & LOQ values (Figure 2).
Based on the response, the established LOD and LOQ values of
Avicel are 0.08 μg/ml and 0.26 μg/ml respectively. The Correlation
coefficient (r) for both LOD and LOQ was found to be 0.999.
Linearity is the ability of the method to produce test results
that are proportional, either directly or by a well-defined
mathematical transformation to the concentration of analyte
in samples within a given range. The method linearity was
demonstrated by preparing solutions over the concentration level
ranging from 2 μg/ml to 4 μg/ml. Linearity graph of concentration
vs absorbance of analyte was plotted. The correlation coefficient
between concentration & absorbance and y-intercept
of the correlation plot was evaluated. The linearity study data is
reported in Table 3 along with the linearity graph (Figure 3). The
method showed a good linearity over the concentration level from
2 μg/ml to 4 μg/ml. The Correlation coefficient (r) was found to
be 1.000 demonstrating the linearity within the specified range.
The system precision was performed by measuring the
standard absorbance in six replicates and calculated % RSD of
results is 0.3. The method precision study was performed by
measuring the absorbance of six different samples of same batch
and calculated % RSD of results is 0.65 (Table 4).
To evaluate the intermediate precision (also known as
Ruggedness) of the method, Precision was performed through
different analyst, on different day by measuring the absorbance
of a six different samples of same sample batch (used in method
precision) and calculated % RSD of results is 0.98 (Table 4).
The ruggedness of method was successfully demonstrated by
computing % RSD of results of twelve samples (six of method
precision and six of intermediate precision) is 0.88 (Table 4).
Pure Avicel API was added in placebo at LOQ, 50%, 100%
and 150% level of sample concentration and prepared spiked
sample solutions as per described method and measured the
absorbance’s of each sample to calculated the recovered amount,
% recovery, mean % recovery and % RSD (Table 5). The recovery
results obtained at LOQ, 50%, 100% and 150% levels are 89.0%,
99.6%, 100.7% and 100.0% respectively with %RSD of recovery
4.45%, 1.87%, 1.87% and 1.71% respectively demonstrating the
good recovery of this method.
The standard solution and sample solution were prepared
as per method described and tested on immediate basis as an
initial result, and then analyzed after every 4 h up to 20 h against
the freshly prepared standard solution and calculated % RSD of
results up to 20 h comparing with initial result. The % RSD of
results for standard solution and sample solution at 20 h are 1.98
and 1.44 (Table 6), this concludes that the Avicel in standard and
sample solution was found to be stable for the period of 20
As part of the Robustness of UV-Visible spectroscopic method,
deliberate change ±3nm in the detection wavelength 635nm
was made to evaluate the impact on the method. To prove the
robustness of analytical method measured the absorbance of
five replicates of standard solution at 632nm and 638nm and
calculated % RSD are 0.33 and 0.16 respectively.
Different APIs and market formulated samples like tablets,
ophthalmic suspensions, nasal spray suspensions was successfully
analysed by this method without any interferences from different
API’s and excipients (Table 7).
The Spray pattern (ovality ratio), Plume geometry (plume
angle), Droplet size distribution and Surface tension measurement
are carried out for the evaluation of impact of Avicel on the nasal
spray suspension product. Accurate determination of Avicel concentration in drug formulation is very critical  since it is
well-known that Avicel addition leads to larger increases in the
formulation viscosity. This in turn affects the surface tension,
spray characteristics and drug deposition pattern of the nasal
spray formulation. The results for one such study with different
Avicel concentration and its impact on critical parameters such
as plume geometry, spray pattern and droplet size in nasal spray
suspension product are reported (Table 8).
Since the deposition pattern is dictated by the droplet size
of the formulation, hence, accurate estimation of Avicel in such
formulations assumes critical importance and our present article
describes the method for accurate estimation of Avicel from
Hence, estimation methods for excipients appear crucial since
any variation in excipients quantity would have a potential impact
on the quality and stability of the developed pharmaceutical
Excipients such as MCC play a significant role in promoting
the manufacturability of drug product, and bioavailability of the
drug substance from the drug product. As a consequence, the
characterization of excipients must go beyond the simple tests
for identity, purity and strength as prescribed in general by the
The different MCC grades greatly impact on the pellet forming
process during the tablet and capsule manufacturing. The
moisture content and shape generally changes during extrusionspheronization
process . The biggest advantage of MCC is its
compatibility with APIs, inertness, ease of handling, and abundant
availability in the market . Recently, we come across one
report published in China on the usage of MCC as an adulterant
in pasteurized milk. The reported method of estimation of MCC
would certainly prove useful in such circumstances.
The uniformity in the chemical and physical characteristics
of any excipient is very crucial in the development of robust and
consistent pharmaceutical products. It is important to minimize
variation between the different batches of excipient, since if there
are significant differences between excipient lots used in clinical
and commercial drug product lots badly affects the bioequivalence
of the drug product. Hence, estimation of excipient concentration
is crucial. Vehovec and co-workers investigated the effect of
inclusion of MCC on the stability of APIs, such as Perindopril
erbumine and Enalapril maleate.
The moisture content of MCC as per European Pharmacopoeia
should not exceed 7.0 % (m/m) since it affects the stability of
moisture-sensitive drugs. The stability of medicinal product is
more with larger particle size of MCC. It is observed that, with
higher content of Avicel, the disintegration time of tablets is
shortened. With lower concentration of MCC, the tablets become
plastic with less hardness. This shows the importance of using
the right concentration of MCC and its accurate estimation from
various pharmaceutical products .
In Biotechnology industry, MCC (Avicel PH-101) has been
successfully used as a delivery carrier of recombinant proteinbased
antigens in animal models . The immune response of the
Avicel absorbed antigen was found to be increased with increasing
Avicel particle concentration, confirming its suitability as a better
immunosorbent for vaccine systems. MCC nanoparticles are found
to be effective in removing organic contaminants and matters
from water . The MCC based nanogels with acrylamide and
acrylic acid are promising adsorbents for the removal of organic
pollutants as well as heavy metals .
Chemical derivatization is a process by which the compound
of interest is converted to compounds with different spectral
properties. One of the primary step of chemical derivatization
includes the use of a derivatizing agents in molar excels of analyte
so that there is complete conversion of the analyte in question.
The derivatized analyte thus formed, have chromophores which
will have absorption in the visible region making its detection
feasible. The process of chemical derivatization can also be applied
to molecules/analytes that have poor absorption in the UV region
(200-280 nm) with significant interference from compounds
that are either impurities or degraded products of compounds
or excipients. Hence a colorimetric method of estimation will
always have advantages over UV/Vis method of estimation using
Accurate determination of Avicel concentration in drug
formulation is very critical  since it is well-known that Avicel
addition leads to larger increases in the formulation viscosity.
This in turn affects the surface tension, spray characteristics
and drug deposition pattern of the nasal spray formulation. The
results for one such study with different Avicel concentration
and its impact on critical parameters such as plume geometry,
spray pattern and droplet size in Nasal spray suspension product.
Since the deposition pattern is dictated by the droplet size of
the formulation, our present article describing the estimation of
method of Avicel in such formulation assumes critical importance
The proposed HPLC method was found to be simple,
specific, precise, accurate, rapid and economical. The developed
chromophore is highly selective with a significantly different lmax
from that of chromogenic agent. UV- Visible spectrophotometry is
simple and sophisticated tools of the analysis. Using diphenylamine
as derivatizing reagent the detection was carried out at 635 nm.
The validated economical method was successfully applied for
forced degradation study of Avicel in different pharmaceutical
formulations and method is found to be selective and stability indicating. Very low concentrations for LOD and LOQ were found
0.08 μg/ ml and 0.26 μg/ ml respectively with linear regression
coefficient of 0.9993. The linearity range of Avicel was proved
from 2-4 μg/ ml with linear regression coefficient of 1.0000. The
% RSD of precision study is less than 2% indicating accuracy and
precision of the method. The mean percentage recovery at LOQ
level is 89% with % RSD less than 5% and at 50%, 100% and
150% levels in between 99-100% with % RSD less than 2%. For
Q1, Q2 sameness study successfully the quantification of Avicel
from different pharmaceutical formulations like tablets, capsules,
nasal suspensions and ophthalmic suspensions etc was performed
by present validated method. The accurate quantitation of Avicel
will help us to specify the important functional tests like spray
pattern, plume geometry and droplet size distribution from
patient point of view. Different grades of Avicel like RC-591,
CL-611, RC 591F are also successfully quantified from different
pharmaceutical formulations. The method can also be extended
for the quantitation of MCC and sodium CMC excipients separately
from different pharmaceutical formulations.
The authors wish to thank Mr. Vinod Ramchandra Jadhav
Chairman and Mr. Avinaash Mandale CEO Sava Healthcare Limited
for their constant support and encouragement. Thanks are also
due to Mr. Sachin Margaj for formatting the figures required for