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Asian Journal of Pharmaceutical Analysis. 10(2): April- June, 2020
67
ISSN 2231–5667 (Print)
2231–5675 (Online)
DOI: 10.5958/2231-5675.2020.00012.5
Vol. 10 | Issue-02|
April- June | 2020
Available online at
www.anvpublication.org
www.asianpharmaonline.org
Asian Journal of Pharmaceutical Analysis
Home page www. ajpaonline.com
RESEARCH ARTICLE
An HPLC Method for detection of Anti-inflammatory Drugs in Bone and
Cartilage health supplements
Sevil Banay Razi1, Farzaaneh Zaaeri2, Hamid Akbari Javar2*
1Department of Drug Quality Assurance, Faculty of Pharmacy, Pharmaceutical Sciences Branch,
Islamic Azad University, Tehran, Iran.
2Department of Pharmaceutics, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran.
*Corresponding Author E-mail: akbarijo@tums.ac.ir
ABSTRACT:
Many bone-building and cartilage repair supplements are available in the market and, many people especially the
elderly people use these supplements. Manufacturers claim various therapeutic properties for these types of
supplements. Therefore, it is likely that these products are included with chemical and anti-inflammatory drugs
that are not listed in labels and brochures. There is not reported using HPLC method to check for chemical and
anti-inflammatory drugs in bone and cartilage supplements, by the competent authorities. In this research, a
valid, simple, affordable and selective method launched to quantify and identify some corticosteroids in bone
and cartilage supplements. This method involves the extraction of the case from supplements using ethanol and
injection directly to high-performance liquid chromatography (HPLC). The effect of different variables such as
mobile phase, column type, pH and other variables was investigated. Optimum conditions including water-
acetonitrile mobile phase with a ratio of 55:45, ODS column, column temperature of 25°C, injection volume of
20 microliters, detector wavelength of 254 nm and the mobile phase flow rate 1ml/min were obtained. With this
HPLC analysis method, cartilage repair products and bone-building supplements can be simply analyzed and the
proportion of infected or free corticosteroids can also be identified.
KEYWORDS: Cartilage repair supplement, Bone-building supplement, corticosteroids, HPLC analysis,
prednisolone, hydrocortisone
INTRODUCTION:
Many bone and cartilage repair supplements are sold in
the market and, elderly people use for improvements in
their bones and cartilages. Producers claim many
therapeutic advantages for these products such as fast
recovery of bone and cartilage. Hence, there is an
uncertainty of prohibited anti-inflammatory and
chemical drugs that are included in products but not
mentioned in labeling.
Received on 12.10.2019 Accepted on 19.11.2019
Accepted on 28.12.2019 ©Asian Pharma Press All Right Reserved
Asian J. Pharm. Ana. 2020; 10(2):67-76.
DOI: 10.5958/2231-5675.2020.00012.5
Food supplements are used for delivery of some essential
food ingredients that are not provided by usual food
regimens. Supplements generally contain vitamins,
minerals, fiber, fatty acids and aminoacids. Food and
Drug Administration (FDA) defines food supplements as
food, although they may be classified as drug or other
products. More than 50,000 kinds of food supplements
are available around the world. More than half of adults
in the united states usually use food supplements
especially multi vitamins[1].
Most of the supplements in the market that claim
fortification and recovery of bones and cartilages or
joints pain reduction, contain Glucosamine and
Asian Journal of Pharmaceutical Analysis. 10(2): April- June, 2020
68
Chondroitin, minerals containing calcium, magnesium,
manganese, zinc, copper, phosphorus, vitamins D, C, K
and fatty acids such as Omega 3. Many elderly people
certified that their pain has decreased after using these
supplements. Therefore, there is a high possibility of
some chemical substances and anti – inflammatory drugs
such as corticosteroids to be included in the composition
of these supplements[1– 4].
Corticosteroids are a group of hormones that act against
inflammation in the body and have anti-inflammatory
properties. These hormones suppress the immune system
to reduce inflammation and pain[2,5]. A lot of drugs based
on endogenous corticosteroids with similar structure and
pharmaceutical action have been built and widely used in
allergy, autoimmune diseases and inflammation.
Examples include betamethasone, dexamethasone,
hydrocortisone, triamcinolone, methyl prednisolone,
prednisone, fludrocortisone, fluticasone, and so on.
These drugs are produced and consumed in the form of
injections, oral drops, ointments and nasal sprays.
Corticosteroid medications are mostly given in the
higher value than the normal amount in the body. Higher
concentrations of these compounds have potent anti-
inflammatory effects and alleviate symptoms of arthritis,
asthma and inflammatory bowel disease[2– 7].
Longtime usage of these pharmaceuticals may increase
blood sugar in susceptible people and leads to
Diabetes[8]. Osteoporosis is a well-known side effect of
long time administration of corticosteroids[9]. Depression
and high blood pressure are other complications. When
patients use corticosteroids, adrenal glands decrease or
stop natural cortisol secretion levels. Therefore,
corticosteroids have many adverse side effects such as
the accession of depression, hallucinations, mania and
manic attacks as well as mental disorders[2]. The effect
on the body's fluid balance and electrolytes, leads to salt
and water retention in the body and ultimately edema[8].
The effect on fat distribution results in the subsequent
accumulation of fat in certain areas of the body such as
back and neck[10]. High blood pressure, high blood
glucose (hyperglycemia), diabetes and hyperlipidemia,
are known as strong factors for cardiovascular disease
and heart attacks[11]. Bone loss and osteoporosis, as well
as rupture or tendon damage in high doses are other side
effects[9,12]. Reduced and weakened layers of the
gastrointestinal mucosa, especially gastric ulcers are
caused and exacerbated[13]. Intraocular high pressure,
glaucoma and cataract are also observed[14]. Therefore,
corticosteroids are usually medicated in combinations
with other safer drugs or as prodrugs in order to release
drugs only near the target site, so that mentioned side
effects are decreased[15– 17].
Products that include combinations of Glucosamine and
Chondroitin make effective help in the treatment of pain
in arthritis. Other applications including the treatment of
rheumatoid arthritis and osteoporosis have not been
proven by research[18]. In the early 1980s, in a cross-
sectional study, 30 hospitalized patients with knee
osteoarthritis were randomized to use placebo, a
combination anti-arthritic medication or glucosamine
sulfate 1500mg daily for three weeks. The group that
received treatment with glucosamine sulfate showed
significant improvements in pain, joint tenderness and
edema, whereas in the control group, there were no such
improvements[19]. The medicinal use of these products is
not approved by FDA (Food and Drug Administration)
and doctors are not allowed to prescribe people as a
medication. Glucosamine and Chondroitinare are often
sold in drug stores as an herbal supplement although
there are no production standards for their preparation as
plant products. Some of these supplements are
containing toxic metals and chemical drugs[20].
The existence of corticosteroids and chemical agents in
food supplements is contrary to regulations of the WHO
(World Health Organization). Accordingly, development
of a method to identify chemical drugs and the quality of
cartilage maker and bone-building supplements is
necessary.
A number of studies have been conducted to evaluate
and analyze combinations of corticosteroids in different
samples such as medications, hair, plasma, urine and
food.UV spectrophotometry, HPTLC (High performance
Thin Layer Chromatography) and HPLC (High
performance Liquid Chromatography) methods have
been used for analysis of corticosteroids[21,22]. Some of
different methods are listed in table 1.
Table1.Example studies in the field of analysis of corticosteroids.
Reference
Sample
Steroid
Instrument/Method
Study
[23]
human urine
Nandrolone
Testosterone
GS / MS extraction by n- pentane
and MSTFA* / NH4I /
Ethanethiol
Identifying and measurement of
anabolic steroids in dietary
supplements
[24]
human plasma
Cortisol, cortisone,
prednisolone, prednisone
LC–MS/MS extraction by
acetonitrile: water: formic acid
(32:68:0.1, v/v/v)
Quantitative analysis of cortisol,
cortisone, prednisolone and
prednisone in human plasma
[25]
human urine
Triamcinolone, cortisone
Prednisone, dexamethasone
methyl testosterone
formoterol
LC-MS / MS extraction by 0.1%
acetic acid in water and
acetonitrile
Detection of corticosteroids,
anabolic steroids and ß2-Agonist
during Athens 2004 olympic games
Asian Journal of Pharmaceutical Analysis. 10(2): April- June, 2020
69
[26]
human urine
prednisolone
prednisolone metabolites
LC-MS / MS extraction by water,
ammonium formate, acetonitrile
with formic acid (0.01%)
Detection and characterization of
prednisolone metabolites in human
urine
[27]
human plasma
methyl prednisolone
LC-MS / MS extraction by
10 mM ammonium formate
buffer and acetonitrile (35:65,
v/v)
Assay of methylprednisolone in
human plasma and its
pharmacokinetic application
[28]
human plasma
human urine
human saliva
prednisone
dexamethasone
cortisone
cortisol
UHPLC-MS / MS extraction by
ammonium acetate in water and
ammonium acetate in methanol
both with 0.1% formic acid.
Determination of glucocorticoids in
human plasma, urine and saliva
[29]
rat plasma
prednisolone acetate
methyl prednisone acetate
LC–ESI MS/MS extraction by
50% water containing 0.01%
formic acid and 50% acetonitrile
A rapid, sensitive and specific
method for the simultaneous
detection of corticosteroids
[30]
human urine
Beclomethasone,
betamethasone
dexamethasone
prednisolone, prednisone
methyl prednisolone
LC-MS / MS extraction by 1 %
Formic acid and acetonitrile
Detection of synthetic
corticosteroids
[31]
human plasma
Prednisolone, prednisone
Cortisol, cortisone
GC–MS derivatization by
heptafluoro-n-butyric anhydride
(HFBA)
Simultaneous determination of
corticosteroids in plasma
*MSTFA: N-methyl-N-trimethyl silyl trifluoro acetamide
**LC–ESI MS MS: Liquid chromatography–electrospray-tandem mass spectrometry
As a result of discussed subjects, specification of a
method for identification of anti-inflammatory and
chemical drugs for quality control of bone and cartilage
fortifier supplements is needed. In this study, we aimed
to practice and validate a standard and simple HPLC
method for identification and determination of chemical
unwanted corticosteroids in bone and cartilage
regeneration supplements. We looked for a separation
method based on gas or liquid chromatography followed
by a spectrophotometric method for specification and
identification of the slightly ingredients. We explained a
standard method using the similar articles as well as
international standards in this field.
MATERIAL AND METHODS:
Full experimental work conducted to set up an accurate,
inexpensive and easy method. All the laboratory
analyses done based on standard Prednisone and
Hydrocortisone.
1. Materials and standards:
Standard prednisone and hydrocortisone were obtained
from Iran hormone industries. HPLC grade methanol and
acetonitrile were purchased from Merck. HPLC grade
purified distilled water was used.
2. Instruments:
High-performance liquid chromatography (HPLC)
system manufactured by DIONEX company with a
gradient pump model Ultimate 3000 equipped with
autosampler 100 microliter injection in maximum was
used. There was a separate oven to adjust the
temperature of the column. UV detector model
UVD170U made by company DIONEX, column C18
(ODS= Octadecylsilane) (460 mm × 250 mm) containing
particles with a diameter of 10µm Manufactured by
Thames Resiek company and the software Auto Chrom
2000 for recording chromatograms were used. UV
spectrophotometer model UV-265FW manufactured by
Shimadzu was used for concentrations evaluation.
Digital scale Model B154 Manufactured by Mettler
Toledo company with a precision of 0.1mg, digital scale
manufactured by Shimadzu with a precision of
0.0001mg, ultrasonic bath model 4200 Manufactured by
Solte company, pH meter manufactured by Mettler
Toledo company and centrifuge model D72 made by
Andreas Hettich company were used. Millipore filtration
system with a vacuum pump and water Purification
system model Basic 360 coupled with the Max-ultra 354
Aqua Manufactured by Younglin company were used to
remove particles from solvents and make HPLC grade
water, respectively.
3. Optimization of the extraction method:
To extract corticosteroids from cartilage repair (bone-
building) supplements, we used solvents that dissolve
these compounds as well but not dissolve other
components. In another word, excipients should be
poorly soluble or insoluble in the solvents. We examined
solvents such as methanol, ethanol, acetonitrile, and
chloroform and investigated the extraction process.
Standard hydrocortisone and prednisolone solutions in a
concentration of 10μg/ml were prepared by each HPLC
grade solvent and centrifuged for 10 min. Supernatants
were diluted 10 times with solvents and their absorptions
were determined by UV spectrophotometer. In the
extraction process, choosing an efficient solvent is the
most crucial part of the process because the solvent must
solve goal substance as well but does not solve other
compounds and excipients in the sample matrix. Since
the compounds found in cartilage maker (bone-building)
supplements are dissolved in water, solvents such as
Asian Journal of Pharmaceutical Analysis. 10(2): April- June, 2020
70
acetonitrile, chloroform, methanol and ethanol were
used. In order to achieve the most appropriate solvent,
extraction process was performed and absorption spectra
were provided.
4. Selection of the optimum condition for HPLC
process:
In this study, we aimed to achieve an optimal method of
high-performance liquid chromatography (HPLC) to
identify and quantify the anti-inflammatory compounds
(hydrocortisone and prednisone) in cartilage maker
supplements. According to previous studies to identify
anti-inflammatory compounds by HPLC method, in
order to view the appropriate peak for these compounds,
the mobile phase of water: methanol in a gradient
washing was used and the percentage of mobile phase
portions was changed from 50:50 (water: methanol) to
100% methanol. Other conditions were set as follows:
C18 column with a length of 12.5cm, a diameter of 4.5
mm and particle size of 10 micrometers, UV detector
wavelength of 254nm, flow rate of 1ml/min for mobile
phase, column temperature of 30oC and injection volume
of 20ml. Standard solutions of prednisolone and
hydrocortisone with a concentration of 0.5μg / ml were
prepared and injected[32–34]. To achieve a fit peak with
the best area in an HPLC analysis, various parameters
including column, type and percentage of mobile phase,
UV detector wavelength, column temperature, injection
volume and flow rate of mobile phase can be changed[35–
38].
To select a proper column, a variety of factors including
properties of substance such as solubility, chemical
nature of the functional groups and molecular weight
should be regarded. It must be examined whether the
species are selectively separable on the stationary phase.
In this study, standard solutions of hydrocortisone and
prednisolone in a concentration of 50μg/ml with C8 and
C18 columns from different companies were studied to
identify anti-inflammatory compounds and acquire the
most appropriate peak. To select a proper column, a
variety of factors including properties of species such as
solubility, the chemical nature of functional groups in
any desired species and molecular weight should be
considered. It must be examined whether the species can
be separated on the selected stationary phase. Chemical
structure of the corticosteroids or anabolic steroids such
as hydrocortisone and prednisone contains a lipophilic
base with four carbon rings and one or more polar
groups resulting being insoluble in water or low soluble.
As a result, column chromatography with C18 stationary
phase and normal phase chromatography are
recommended to identify and isolate them. Columns
have large differences in retention of materials due to the
different construction of active silanol groups.
Mixtures of water and acetonitrile are the most common
mobile phases for liquid chromatography with high
efficiency. Acetonitrile and methanol have different
chromatographic characteristics. Acetonitrile has low
viscosity, low UV absorption, good kinetics which
makes sharper peaks, more efficient washing that leads
to lower consumption of solvent, and less back pressure
for acetonitrile-water mixtures in comparison with
mixtures of methanol-water. Methanol is odorless and
less toxic than acetonitrile which causes better and safer
working conditions. Salts have better solubility in it and
the risk of deposit formation is lower. It is suitable for
isolation of bases in alkaline pH condition. On the other
hand, the polarity of the mobile phase is effective in
separation of species and chromatographic washing. In
reverse phase systems with a non-polar C18 stationary
phase, non-polar species have longer retention times and
their exit time from the column is altered by changing
the ratio of mobile phase. Among three solvents of
water, acetonitrile and methanol, dipole moment in water
is stronger and as a result water has the highest and
methanol has the lowest polarity. But water is not a good
washing solvent for corticosteroids causing delayed
elution, so the percentage of organic solvent should be
increased.
Changes in column temperature cause changes in the
chromatographic parameters (viscosity, kinetics,
efficiency) and chemical or thermodynamic changes
(enthalpy of adsorption, retention time and selectivity).
An increase in temperature from 30°C to 40°C causes
peaks move at different speeds toward the beginning of
the chromatogram and the retention time and selectivity
is slightly reduced but the column pressure is increased.
In this study, standard solutions of hydrocortisone in
concentration of 100μg/ml and prednisolone in
concentration of 20μg/ml were prepared and different
temperatures from 25 to 40°C were applied on the
column. The peaks obtained and the effect of
temperature on the area under the peak and retention
time of samples was analyzed.
Ideal flow rate for this study was considered 1 ml/min.
To confirm method robustness and the optimal flow rate,
flow rates from 0.9 to 1.1ml/min using standard
solutions of hydrocortisone 100μg/ml and prednisone
20μg/ml were applied. Each standard solution was
individually injected three times to the HPLC system and
the effect of flow rate on the retention time and the area
under the peak of standard solutions and control samples
was studied. According to the conducted review on
chromatograms, applying the flow rate of 1/1 ml/ min for
both standard and control samples reduces retention time
of the samples; the sample and the solvent peaks may
interfere, and the area under the peak may show a
decline. While applying a flow rate of 0.9ml/min for
both solutions increases the retention time of the sample
Asian Journal of Pharmaceutical Analysis. 10(2): April- June, 2020
71
there by the sample is removed later and the area under
the peak is also enlarged.
To confirm an appropriate wavelength, wavelengths of
250nm and 260nm using standard solutions were applied
and retention time and area under the peak of each
method were studied.
In order to perform quantitative analysis and
measurement of anti-inflammatory compounds in these
supplements, calibration curves for each compound was
separately plotted. To plot calibration curve of
hydrocortisone, solutions with different concentrations
from 10 to 130μg/ml were prepared and the best
concentration range for plotting the curve was obtained.
The appropriate graph with concentrations in the range
of 70 to 130μg/ml was plotted. 5 solutions at
concentrations of 70, 80, 130, 115, 110μg/ml were
injected into the HPLC system. Each sample was
analyzed three times and a calibration curve was plotted.
To plot calibration curve of prednisolone, solutions with
different concentrations from 1 to 13μg/ml were
prepared and the best concentration range was obtained.
The appropriate graph with concentrations in the range
of 7 to 13μg/ml was plotted. 5 solutions at
concentrations of 7, 9, 10, 12, 13μg/ml were prepared
and injected into the HPLC system. Each sample was
analyzed three times and a calibration curve was plotted.
5. Method Validation:
After the best analysis method was achieved, it should be
validated by the conditions and standard materials.
Validation methods were applied to investigate
validation parameters for evaluation of hydrocortisone
and prednisolone in bone-building and cartilage repair
supplements.Validation factors including selectivity,
linearity, precision, accuracy and limit of detection were
measured[32–40].
Linearity within the range of analysis should be
confirmed. Linearity is determined by visual inspection
of calibration curve plotted based on analytical signals
against samples concentrations. After the initial ensuring
linearity of the graph, a closer look should be done by an
appropriate statistical method. To evaluate the linearity
of hydrocortisone analysis method, standard solutions
with concentrations of 70, 85, 90, 100, 110, 115 and
130μg/ml were prepared and each solution was injected
twice into the HPLC system and linearity curve was
plotted. For prednisolone the method was conducted in
the same way, but with different concentrations of 7, 8,
9, 10, 11, 12 and 13μg/ml[35].
Precision of an analysis method indicates the degree of
coordination among the results of separate tests in
condition that multiple samples are taken from a
homogeneous sample. Precision of an analysis method is
usually explained by standard deviation (SD) factor and
relative standard deviation (RSD) that is also stated as
coefficient of variation (CV). Precision may be
illustrated as reproducible precision or reproducibility of
the method of measurement under normal conditions of
analysis. Precision of the analysis method is determined
at two levels. Repeatability was specified by
determination of technique precision in one day, in one
laboratory and by one examiner. This way is also called
Intra-day evaluation of precision. Reproducibility was
elucidated by measurement of precision in different
days, in different laboratories by different devices and
testers which is also called the Inter-day precision.
Precision of the method was carefully specified for
hydrocortisone and prednisolone analysis by HPLC.
The real amount of active ingredient in a pharmaceutical
formulated product is nominated as accuracy. Accuracy
is verified by adding a known amount of substance to the
excipient materials in the range of analysis method
which can be precisely determined. If it is not possible to
provide samples of the excipients, another acceptable
way to determine accuracy is that known amounts of
substance is added to pharmaceutical product and the
results of analysis of substance is compared with the
results of another proposed method that its accuracy has
been proven. Hence for the determination of accuracy of
the method, standard solutions of hydrocortisone with
concentrations of 90 and 110μg/ml and prednisolone
with concentrations of 8 and 11μg/ml were added to the
samples and injected 3 times into the HPLC system. The
recovery for each concentration was calculated and
reported as accuracy.
According to ICH guidelines, signals measured with low
concentrations of certain species should be compared
with blank samples (control). The lowest concentration
of the sample which is detected and differentiated from
background noise (the blank), is introduced as detection
limit. Signal to noise ratio of 3: 1 or 2: 1 is accepted.
Detection limit is not involved in quantification of
samples. (LOD =Signal /noise)
To determine the quantification limit of method,
according to ICH guidelines, signals for low
concentrations of samples should be compared with
blank samples (control). The lowest concentration which
is reliably determined, is introduced as limit of
quantification. Signal to noise ratio of 10: 1 is considered
as LOQ that enables reasonably recognition of the
concentrations. (LOQ = Signal /noise)
Proof of selectivity or specificity requires in determining
the amount of impurities with appropriate accuracy and
precision along showing that the process of analysis is
not affected by the presence of impurities. In practice,
this can be achieved by adding raw pharmaceutical
material or product to a mixture of appropriate amounts
Asian Journal of Pharmaceutical Analysis. 10(2): April- June, 2020
72
of impurities and excipients showing that the results of
the quantification are not influenced by the presence of
foreign substances. Selectivity of the method for
determination of hydrocortisone and prednisolone was
evaluated by adding 1ml of 5μg/ml standard solution of
hydrocortisone to 100mg of product powder which is
free of any anti-inflammatory drug. Methanol was added
to bring the volume and the mixture was stirred to be
completely uniform. Then, it was centrifuged for an hour
and the supernatant was tested. This method was
similarly done for prednisolone[35,36].
Three types of powdered cartilage supplements in the
market were tested. 1 g of each sample was extracted
about drug by solvents. The resulting solution was
injected directly into the HPLC system.
RESULTS AND DISCUSSION:
Differences in extractions by the four solvents
represented that both chloroform and acetonitrile do not
show acceptable separated spectra for substances.
Ethanol and methanol both are suitable solvents to
extract both substances but ethanol as a clean, cheap and
safe solvent is preferred (fig.1).
Figure1. The absorption spectra of prednisolone and hydrocortisone
extracted by different solvents: prednisolone extracted by
acetonitrile(A), chloroform(B), methanol(C), ethanol(D) and
hydrocortisone extracted by acetonitrile(E), chloroform(F),
methanol(G), ethanol (H).
The column (C18, 250mm, 4.5mm and 10μm) from
Thames Restek Ltd UK was used to find optimum
conditions. According to previous studies, water-
methanol mobile phase in gradient mode was used at
first. Prednisolone and hydrocortisone peaks were
appeared separately in this condition. Since the peaks
had low height, the reproducibility of results was not
observed in subsequent injections declaring that this type
of mobile phase is not appropriate to identify
prednisolone and hydrocortisone by this method[36,37]. At
this stage, due to the chemical nature of species and
instrumental conditions, the mobile phase mixture was
changed to water: acetonitrile. Therefore, different
percentages of the two solvents were studied and ratios
of 45:55 and 55:45 (reducing the amount of acetonitrile
and increasing the percentage of water) were analyzed. It
was observed that by increasing the percentage of
aqueous solvent, the retention time for hydrocortisone
and prednisolone increases; while by reducing the
percentage of organic solvent, the retention time, the
area under the peak and the resolution of peaks are
optimal. Thus 45% of acetonitrile according to tests was
carried out. Chromatograms related to the ratios of
mobile phase for prednisolone and hydrocortisone
standards are showed in figure 2. The best ratio of the
two solvents, according to the parameters of retention
time and peak area was selected 55:45 (water:
acetonitrile).
Figure2. Standard chromatogram of prednisolone and hydrocortisone
in different ratios of solvents: Standard chromatogram of prednisolone
in mobile phase with the ratio of water: acetonitrile 65: 35(A),
Standard chromatogram of hydrocortisone in mobile phase with the
ratio of water: acetonitrile 65: 35(B), standard chromatogram of
prednisolone in mobile phase with the ratio of water: acetonitrile 55:
45(C), standard chromatogram of hydrocortisone in mobile phase with
the ratio of water: acetonitrile 55: 45(D).
As regards a decrease in retention time depends on the
sample and the mobile phase, the results for effect of
rising the temperature on retention time of
hydrocortisone and prednisolone standards showed that
the best column temperature for analyzing the samples is
30 °C. The effect of rising the column temperature was
Asian Journal of Pharmaceutical Analysis. 10(2): April- June, 2020
73
also tested on the area under the peak and it was found
that the column temperature has no effect.
Chromatograms of standard concentrations of drugs in
30°C column temperature are shown in figure 3.
Figure3. Chromatograms of prednisone and hydrocortisone standards
in different column temperatures: Chromatograms of prednisolone
standard in column temperature of 30°C (A), Chromatograms of
hydrocortisone standard in column temperature of 30°C (B),
Chromatograms of prednisolone standard in column temperature of
40°C (C), Chromatograms of hydrocortisone standard in column
temperature of 40°C (D).
As a result of the study of retention time and acceptable
peak shape, optimum flow rate was determined 1ml/min.
Average area under the peak and retention times for
drugs are depicted in table 2.
Table2.Evaluation of retention time and area under the peak for
drugs in different flow rates
Area under the peak
Retention time
(min)
0.9
1
1.1
0.9
1
1.1
Flow rate
(ml/min)
27.32
25.26
10.88
4.37
3.28
3.55
Prednisolone
75.06
69.52
61.33
9.65
6.52
7.55
Hydro
-cortisone
Maximum absorption for prednisolone and
hydrocortisone was observed at about 250 nm. Using
UV detector in wavelengths of 250 and 260 nm for
detection of hydrocortisone and prednisone standards
showed similar reduction in the area under the peak than
254 nm. According to the results (table 3), a wavelength
of 254 nm is desirable because the retention time and
area under the peak are desirable and there is no
interference between sample and solvent.
Table3. Evaluation of retention time and area under the peak for
drugs in different Wavelengths
Area under the peak
Retention time
(min)
260
254
250
260
254
250
Wavelength
(nm)
342.5
518.7
285.3
4.3
3.9
4.2
Prednisolone
581.1
654.6
521.1
8.9
8.4
9.1
Hydro
-cortisone
Standard calibration curve of prednisolone was plotted
using standard solutions in the range of 7 to 13μg /ml.
Standard calibration curve of hydrocortisone was plotted
using standard solutions in the range of 70 to 130μg/ml.
The manner and linearity of the calibration curves with
the deviation of points of R> 0.99 represents a linear
correlation for the method in used concentrations (fig.4).
Figure4. Calibration curve and linearity evaluation for prednisolone
(A) and hydrocortisone (B) assay
Standard solutions of hydrocortisone and prednisolone
were injected into HPLC system to study precision of
within day (intra-day) and between days (inter-day)
working. The results were arranged in table4.
Asian Journal of Pharmaceutical Analysis. 10(2): April- June, 2020
74
Table 4. Results of within day and between day precision study for prednisolone and hydrocortisone: intra-day precision study for
prednisolone (A), hydrocortisone (B), inter-day precision study for prednisolone (C), hydrocortisone (D)
Concentration
(µg/ml)
AUC
Sample 1
AUC
Sample 2
AUC
Sample 3
RSD%
Sample 1
Sample 2
Sample 3
20
25.290
24.381
24.535
First
day
0.074
0.069
0.038
20
25.288
24.319
24.695
Second day
0.092
0.058
0.032
20
25.257
24.407
24.696
Third day
0.083
0.063
0.023
Mean
25.278
24.369
24.642
Mean
RSD%
0.083
0.063
0.031
SD
0.019
0.168
0.093
RSD%
0.074
0.689
0.377
A
C
B
D
Concentration
(µg/ml)
AUC
Sample 1
AUC
Sample 2
AUC
Sample 3
RSD%
Sample 1
Sample 2
Sample 3
100
69.721
72.282
69.463
First day
0.023
0.090
0.058
100
69.393
72.423
69.545
Second day
0.031
0.095
0.060
100
69.140
72.374
69.502
Third day
0.027
0.011
0.062
Mean
69.711
72.354
69.503
Mean
RSD%
0.027
0.069
0.060
SD
0.016
0.066
0.041
RSD%
0.023
0.090
0.058
Standard solutions of two drugs with different
concentrations in the linear range were injected into the
HPLC system. Accuracy was determined by the recovery
percentage of species. The obtained results were located
in the range of 97% -103% (table 5).
Table 5. Results of accuracy verification for prednisolone (A) and
hydrocortisone (B) standards
Concentration
(µg/ml)
A
Recovery percent
(% R)
Peak area
Mean
8
38.630
38.599
100.07
8
38.658
8
38.511
11
55.259
55.353
101.05
11
55.488
11
55.277
Concentration
(µg/ml)
B
Recovery percent
(% R)
Peak area
Mean
90
39.292
39.158
102.09
90
39.129
90
39.054
110
47.451
47.635
100.01
110
47.569
110
47.885
The lowest concentrations of standard solutions of
prednisolone and hydrocortisone were identified and
detected equal to 0.001μg/ml and 0.02μg/ml,
respectively. Signal to noise ratios for prednisolone and
hydrocortisone in mentioned concentrations were
calculated 5.55 and 3.09, respectively, that are in the
acceptable range of ≥ 3 and ≤ 10. The lowest
concentrations of prednisolone and hydrocortisone
standards were measured equal to 0.01μg/ml and
0.2μg/ml, respectively. Signal to noise ratios for
prednisolone and hydrocortisone in mentioned
concentrations were calculated 12 and 16, respectively,
that are in the acceptable range of ≥ 10.
Solutions resulted from the extraction processes were
injected into the HPLC system, and at wavelength of
254nm two peaks related to prednisolone and
hydrocortisone were appeared. Chromatograms obtained
from analyzing standard solutions of hydrocortisone and
prednisolone, cartilage supplements without
hydrocortisone and prednisolone (placebo) and cartilage
supplements containing hydrocortisone and prednisolone
(Spiked) at the wavelength of 254nm showed that the
two drugs can be separated by a resolution coefficient of
>3 and do not interfere with other ingredients (fig.5).
Figure 5. Results for analyzing supplements at 254 nm to study
specificity of the method: standard solution of prednisolone (A),
cartilage supplement without prednisolone (B), cartilage supplement
containing prednisolone (C), standard solution of hydrocortisone (D),
cartilage supplement without hydrocortisone (E), cartilage supplement
containing hydrocortisone (F).
Asian Journal of Pharmaceutical Analysis. 10(2): April- June, 2020
75
Analysis of real samples of cartilage supplements
(bone-building supplements):
Three cartilage repair supplements were analyzed by
described HPLC procedure at the wavelength of 254nm
to examine whether the claims on the labels of these
supplements that are free of any chemical compounds,
are true or not. Chromatograms derived from the
supplements at 254nm are shown in Figure 6.
Figure 6. Chromatograms obtained from the analysis of real samples
of cartilage repair supplements in the market (Glucosamine capsules)
for prednisolone and hydrocortisone
According to the chromatograms, to determine the
amount of active ingredients of prednisolone and
hydrocortisone in three glucosamine capsule samples,
the average area under the curve of prednisolone and
hydrocortisone in real sample solutions, were determined
and the amount of the compounds found in each sample
was calculated using calibration line equation and
reported in µg/g value (table 6).
Table 6. The results for assay of prednisolone and hydrocortisone
in three samples of glucosamine capsule in the market
Corticosteroid
Sample A
(µg/g)
Sample B
(µg/g)
Sample C
(µg/g)
Prednisolone
0.27
0
0.25
Hydrocortisone
2.37
0.59
1.04
This study aimed to achieve an optimal HPLC method to
identify and quantify the anti-inflammatory compounds
(prednisolone and hydrocortisone) in cartilage repair
products. According to research done, parameters such
as column type, the wavelength of detector, the
percentage of each solvent in mobile phase, column
temperature, flow rate and injection volume for the
analysis of prednisolone and hydrocortisone were
optimized. Optimum conditions of parameters are
presented in the table 7.
Table 7. Optimum values for analysis of prednisone and
hydrocortisone by HPLC method
Optimized condition
Parameters influencing in the
analysis
C18: 25cm/4.5 mm/10µm
Column: length/diameter/stationary
phase particles diameter
254 nm
Wave length of detector
water: acetonitrile 55: 45
Mobile phase
30oC
Column temperature
1 ml/min
Flow rate
20 µl
Injection volume
CONCLUSION:
In this study an HPLC analysis method for determination
of the amount of corticosteroids (prednisolone and
hydrocortisone) in cartilage repair supplements was
tested. The method is simple, rapid, selective, affordable
and valid. The method of extraction and sample
preparation are simple and fast as well as the method is
highly efficient. The method showed acceptable
accuracy and precision in an appropriate linearity range
therewith low detection and quantification limits, for the
analysis of corticosteroids in cartilage repair
supplements. In this method, the matrix of supplements
(excipients) does not interfere to the evaluation of
corticosteroids. The developed method was accepted as a
valid applicable instruction for evaluation of
corticosteroids in cartilage repair products and validated
as a Standard Operating Procedure (SOP) by Iran
Reference Food and Drug Control Laboratories. As a
result, evaluation of cartilage repair (bone-building)
supplements that are imported to the country and
estimation of products infected with corticosteroids is
accomplished by this method. This work is the first study
done toward the assessment of dietary cartilage repair
(bone-building) supplements regarding to the
determination of corticosteroids.
ACKNOWLEDGEMENT:
This work was supported by Iran Food and Drug Control
Laboratories by the equipment and materials but it did
not receive any grant from funding agencies in the
public, commercial, or not-for-profit sectors.
CONFLICT OF INTEREST:
The authors announce that there is no conflict of interest
about article.
Asian Journal of Pharmaceutical Analysis. 10(2): April- June, 2020
76
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