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Submission: February 06, 2019; Published: February 21, 2019
*Corresponding author: Mohamed EM Hassouna, Department of Chemistry, Faculty of Science, Beni-Suef University, Egypt
How to cite this article: Mohamed E M Hassouna , Mahmoud A Mohamed. Novel RP-HPLC-DAD and RP-UPLC Methods for Simultaneous Determination
of Cefaclor and Methylparaben in their Dosage form and in its Impurity Cefaclor -Delta-3- Isomer. Glob J Oto, 2019; 19(3): 556014. DOI: 10.19080/GJO.2019.19.556014
Cefaclor (CFC), a new orally administered for the second generation of cephalosporin is the most effective for treatment of acute otitis media in children. So, the main task of this work is to develop and validate a novel RP-HPLC and RP-UPLC methods for simultaneous determination of cefaclor and methylparaben (MP) in their powder for oral suspension (POS) dosage form and in its impurity cefaclor-delta-3- isomer (CD3I). The chromatographic system is performed at ambient temperature using mobile phase composing of acetonitrile: methanol: 0.02M ammonium dihydrogen phosphate pH 4.7 ± 0.1 (25:10:65, v/v) on Agilent Eclipse XDB C18 column (250 mm X 4.6 mm, 5 μm particle size) at flow rate 1.0 mL/min, injection volume 20 μL for RP-HPLC and Waters CORTECS® C18 column (50 mm × 4.6 mm, 2.7 μm particle size) at flow rate 0.3 mL/min, injection volume 0.2 μL for RP-UPLC and UV detection at 265 nm. The method is linear for all analytes in the concentration range (70-700) μg/mL for CFC and (10-200) μg/mL for MP with correlation coefficients >0.999. The suggested method is validated according to ICH guidelines. Hence it is suitable for laboratory control of raw materials, bulk and finished products.
Cefaclor (CFC) therapy is very effective for treating children aged 2-6 years affected by acute otitis media, with no significant difference related to treatment duration. Short-course treatments appear better in terms of compliance and tolerability . Both cefaclor and amoxy-clav caused a significant improvement in all the signs and symptoms in the treatment of acute otitis media after 10-days treatment period. However, between-the-group comparisons showed that the lowering in most of the symptoms was significantly more in cefaclor drug than amoxicillin-clav . CFC, (Figure 1a) is a semisynthetic product derived from a fermentation product follows the second generation of cephalosporin antibacterial group for oral administration. It is chemically named as (6R,7R)-7-[[(2R)-2-Amino-2-phenylacetyl] amino]-3-chloro-8-oxo-5-thia-1-azabicyclo [4.2.0] oct-2-ene-2-carboxylic acid monohydrate. It has a molecular formula of C15H14ClN3O4S, H2O and a molecular weight of 385.8. It has characters of appearance; White or slightly yellow powder and has solubility as slightly soluble in water, practically insoluble in methanol and in methylene chloride . CFC is indicated for the treatment of the following types of infections caused by or likely to be caused by susceptible organisms: lower respiratory
infections, including pneumonia, bronchitis, and exacerbations of chronic bronchitis. Also, upper respiratory infections, including pharyngitis, tonsillitis and otitis media. Skin and skin structure infections. Urinary tract infections, including pyelonephritis and cystitis.
In more serious infections, otitis media, and infections caused by less susceptible organisms, the recommended dosage is 40 mg/kg/day in divided doses every 8 to 12 hours (maximum 2 g/day). For otitis media, 12 hourly administration appears equally effective . MP (Figure 1b), is used as antimicrobial preservative. It was chemically named as (methyl 4-hydroxybenzoate), a molecular formula of (C8H8O3) and a molecular weight of 152.1. It has characters of appearance white or almost white, crystalline powder or colorless crystals and has characteristic of solubility as very slightly soluble in water, freely soluble in ethanol (96 per cent) and in methanol . Cefaclor -delta-3- isomer (CD3I), (Figure 1a) is used as antibacterial broad-spectrum drug. It was chemically named as (2R,6R,7R)- and (2S,6R,7R)-7-[[(2R)-2-amino-2-phenylacetyl] amino]-3-chloro-8- oxo-5-thia- 1-azabicyclo [4.2.0] oct-3-ene-2-carboxylic acid (delta-3-cefaclor). It has a molecular formula of C15H14ClN3O4S, and a molecular weight of 367.8. It has characters
of appearance: white powder and has solubility as slightly
soluble in water .
HPLC method for determination of CFC is officially reported
in both of British Pharmacopeia (BP) and United States
Pharmacopeia (USP) . It is still a limited number of analytical
methods that are reported for the determination of CFC and MP
individually or in combination with other drugs including high
performance liquid chromatography (HPLC) [6-16], capillary
electrophoresis and electrochemical methods [17-19], thin layer
chromatography (TLC) [20-22], UPLC and mass spectrometry
, kinetics degradation and spectrophotometric [24-31], and
comparative in vitro dissolution studies [32-34]. For the best of
our knowledge, there is no RP-HPLC and RP-UPLC methods for
simultaneous determination of cefaclor and methylparaben in
their dosage form and in its impurity (cefaclor -delta-3- isomer).
So, it is thought worthwhile to develop and validate a simple,
precise and accurate RP-HPLC and RP-UPLC methods to resolve
this binary mixture and determine cefaclor -delta-3-isomer in
their dosage form.
CFC and MP are kindly supplied by Hikma pharmaceutical
industries company, Beni-Suef, Egypt with potency >99.0%.
Cefaclor-delta-3-isomer is purchased from store of USP with lot
no. (R044L0). Alfatil® 125 mg/5mL “POS” (Batch No. 0004D4)
is manufactured by Laboratories Ethypharm, France.
Agilent 1260 infinity (Germany) HPLC system is supplied
with preparative auto sampler (G1329B), Quaternary pump
(G1311C), Thermostatic column compartment (G1316A), DAD
detector VL (G1315D) and ChemStation (chem32) Software.
Waters ® UPLC System, a quaternary liquid chromatography
provides plug with 2489 UV/Vis Detector (A17VTU408A),
Sample manager FTN-R (D17VSM260N), Quaternary solvent
manager-R (C17VQS618G) and Empower™ 3 Software. UV- 1800
double beam UV-Visible spectrophotometer (Shimadzu-Japan)
with the highest resolution and spectral bandwidth of (1nm from
190-1100 nm range) is used for all absorbance measurements.It is matched with 1 cm quartz cells. Perform data analysis by
software (UV-Probe 2.3.3). METTLER TOLEDO density meter.
Stock standard solution (1000 μg/mL of CFC): Carefully
transfer 100 mg of CFC into 100 mL volumetric flask, add about
70 mL of the solvent. Sonicate for 10 min till complete dissolution
then completes to mark with the same solvent and mix well.
Stock standard solution (1000 μg/mL of MP): Similarly
weigh 100 mg of MP into 100 mL volumetric flask, add about 70
mL of the solvent Sonicate for 10 min till complete dissolution
then completes to mark with the same solvent and mix well.
Standard solution (625 μg/mL) of CFC and (150 μg/mL)
of MP: Accurately transfer 12.5 mL aliquot from CFC and 3mL of
MP the previously prepared stock standard solutions into 20-mL
volumetric flasks. Complete to mark with the same solvent. Mix well and filter through 0.45 μm PTFE membrane filter and inject
into the chromatographic system (Figure 2).
Take one bottle, add the quantity of water as mentioned in
the product label. Close the bottle and shake well for 10 minutes.
Use suitable calibrated analytical balance, weigh an empty
clean and dry pycnometer. Fill the pycnometer completely with
water and weigh. Then repeat filling it with the reconstituted
suspension and weigh and determine the specific gravity
(density) according to the following equation:
Constitute bottle of Alfatil ®125 mg /5 mL POS as directed
in the label and shake well for 10 minutes using multi shaker.
Use the previously determined specific gravity to calculate
the required weight per grams for each test as the following
Transfer an accurately weighed portion of the resulting
suspension, freshly mixed and free from air bubbles, dilute
quantitatively with solvent in 200-mL volumetric flask to obtain
a final solution (equivalent to about 125 mg of CFC and 30 mg
MP). Sonicate if necessary, to ensure complete dissolution of the
CFC. Filter through 0.45 μm PTFE membrane filter and inject
into the chromatographic system.
System suitability solution: dissolve a quantity of USP
cefaclor, delta-3 isomer RS in the standard solution to obtain a
solution having a concentration of about 0.05 mg per mL and
filter through 0.45 μm PTFE membrane filter and inject into
the chromatographic system (Figure 3). Chromatograms using
solvent as blank. Test solutions: constitute a bottle of Alfatil
®125 mg /5 mL POS as directed in the label and shake well for
10 minutes using multi shaker. Transfer an accurately weighed
portion of sample, freshly mixed and free from air bubbles,
equivalent to about 50 mg of cefaclor (about 1.9066 gm), to
10-mL volumetric flasks, dilute each with solvent to volume,
and mix. Sonicate briefly if necessary, avoid heating and filter
through 0.45 μm PTFE membrane filter and inject into the
chromatographic system (Figure 4).
Separately transfer different aliquots from the stock standard
solutions into separate series of 10 mL volumetric flasks covering
the concentration ranges (70-700) μg/mL for CFC and (10-200)
μg/mL for MP. Triplicate 20 μL injections are injected for each
concentration applying the recommended chromatographic
conditions. The chromatograms are recorded, and the peak
areas of CFC and MP are estimated, the calibration curves
relating the obtained integrated peak areas to the corresponding
concentrations are constructed and the regression equations are
Many systems of different compositions and ratios are tried
including organic: methanol (100%); methanol: purified water
(50:50, v/v) and acetonitrile: purified water (50:50, v/v). Also
(0.2 to 2.0 mL/min) flow rates are tried on HPLC and UPLC
system, to gain the optimum wavelength for the solution, 20
μg/mL of CFC and 25 μg/mL of MP are measured at 200 to 400
nm (Figure 5). Thus, 265 nm was selected as the most suitable
wavelength. Preliminary studies involved trying C18 reversed phase columns. The best developing system using mobile
phase composing of acetonitrile: methanol: 0.02M ammonium
dihydrogen phosphate pH 4.7 ± 0.1 (25:10:65, v/v) on Agilent
Eclipse XDB C18 column (250 mm X 4.6 mm, 5 μm particle size)
at flow rate 1.0 mL/min, injection volume 20 μL for RP-HPLC
and Waters CORTECS® C18 column (50 mm × 4.6 mm, 2.7 μm
particle size) at flow rate 0.3 mL/min, injection volume 0.2 μL
for RP-UPLC and UV detection at 265 nm. Another advantage
for UPLC system is the relatively short run time (6min) of the
analysis, under isocratic conditions. This selected developing
system allows good separation with good Rt values without
tailing of the separated bands and good theoretical plates.
The linearity of the suggested methods is obtained in the
concentration range (70 – 700 μg/mL) for CFC and (10 – 200 μg/
mL) for MP. From the graphical presentation, the relation of peak
response (A) plotted at X axis and concentration (C) plotted at
Y axis is evaluated on the base of least-square linear regression
equation (A= slope C + Y intercept). The LOD and LOQ of the
method are calculated using the expression (3.3σ/ slope) and
(10σ/slope) respectively. The obtained coefficient of regression
is >0.999. Linearity results are shown in (Table 3).
Repeatability: repeatability is proceeded using 6 replicates
of the standard solution of the compound being studied (625
μg/mL) for CFC and (150 μg/mL) for MP. The system is precise
as the relative standard deviation RSD ≤ 2%. High precision
of the method is obtained as shown in (Table 3). Intermediate
precision (ruggedness): Intermediate precision expresses the
within-laboratories variations: different days, different analysts,
different equipment’s, etc. Good results are obtained and
presented in (Table 5).
Robustness: for measuring the capability of the method
to remain unaffected by small, but deliberate, variations in
method’s parameters such as: influence of variations of flow rate change (±0.1 mL/min), wave length change (265 ± 2.0 nm),
column temperature change (25±5 ˚C), and pH of the mobile
phase (±0.2). Good results are gained as presented in (Table 5).
Stability of the analytical solution: After preparing the
standard solution, part of it was stored in fridge and another
one is stored at room temperature. After about 24 hours, these
solutions are tested against freshly prepared standard; relative
standard deviation should be less than 2%. These results are
displayed in (Table 5).
The suggested method is performed by assaying six
samples from the finished product with the same concentration
(Alfatil®125 mg /5 mL POS) to determine the assay of CFC and
MP (Figure 6). The obtained results are displayed in (Table 6).
System suitability testing is an integral part of much
analytical procedure. The tests are based on the concept that the
equipment’s, electronics, analytical operations and samples to
be analyzed constitute an integral system that can be evaluated
as such. System suitability is checked by calculating the tailing
factor (T), column efficiency (N), resolution (Rs) factors. All
calculated parameters are found to be within the acceptable
limits indicating good selectivity of the method and assuring
system performance (Table 7).
Method selectivity is established for the finished product
being studied and placebos matrix containing all excipients of the
dosage form. The results referred that no interference is detected
at the retention times of CFC and MP in placebo solution. Also,
the standard addition technique is applied by adding known
concentrations of pure form for both drugs to dosage form and
the recovery of the added pure forms are calculated to check the
specificity and selectivity of the method. The obtained results
are shown in (Table 6).
Accelerated stability study intended to increase the rate of
physical changes as (appearance, pH, water content…etc.) or
chemical degradation (assay, dissolution, related substances and
so on) by using immoderate conditions of elevated temperature
and humidity. So, stability study is performed under storage
conditions (40 ±2oC / 75 ±5 % RH (relative humidity) for six
months. Products are recommended to analysis at three points
(e.g. 0, 3 and 6 months). The obtained results shown in (Table 8).
A novel, accurate and sensitive RP-HPLC-DAD and RPUPLC
methods were validated and developed according to
the requirements of ICH guidelines for the simultaneous
determination of cefaclor and methylparaben in their dosage
form and in its impurity (cefaclor-delta-3- isomer). Based on the
above results, the analytical method is valid, achieved reduced
time for analysis of assay & related compound and can be used
for regular routine quality control analysis and stability study.