|
Available online from February 02, 2026
Asian Journal of Pharmaceutical Analysis.
2026; 16(1):21-28.
|
|
This work is
licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0
International License. Creative Commons License.
|
|
1. INTRODUCTION:
Tuberculosis (TB) is still a serious
worldwide concern, the which has becoming more difficult to control since
Multidrug-resistant (MDR) and extensively Mycobacterium tuberculosis types that
are highly resistant to drugs Sis. An anticipated 10.4 million new 480,000 new
cases and (incident) TB cases globally of TB that is resistant to several drugs
(MDR-TB). An estimate was 1.4 million TB deaths occurred in 2015, and it
continued to be one Among the top ten global causes of death1. Present
Treatment for tuberculosis is ineffective because of several factors, such as
extended treatment duration, hepato- toxicity and other adverse effects of
traditional medications, and patients’ early self-termination2.
Consequently, Combination treatment is essential for lowering the likelihood of
resistance by reducing the length of treatment, Making the availability of new
drug combinations a priority necessity.3
Streptomyces Mediterranean is the source of rifampicin
(RIF), a complex semisynthetic macrocyclic rifamycin class of antibiotic used
to treat tuberculosis and other infectious diseases4–7. It
is classified as a first-line antituberculosis medication. RIF suppresses RNA
synthesis and causes cell death by inhibiting DNA-dependent RNA polymerase. It
is limited to treating mycobacterial infections, where the traditional use of
combination medications delays the development of resistance, and the treatment
of asymptomatic meningococcal carriers due to the quick formation of resistant
bacteria8.
Second- or third-line medications, such as
levofloxacin (Lev), are frequently used to treat resistant forms of
tuberculosis. Lev is a member of the broad-spectrum antibacterial medication
class known as third-generation fluoroquinolones. Levofloxacin is therefore
less effective when used alone than when combined with other medications9.
Levofloxacin’s minimum inhibitory concentration (MIC) for M. tuberculosis is
0.25–4 mg/L10.
Drugs are incorporated into a variety of delivery
systems, such as liposomes, β-cyclodextrin derivatives, polymers, etc., to
improve treatment efficacy and minimize side effects. Inhalation is one of the
most promising methods of delivering such systems for antituberculosis therapy
because it allows the drug formulation to efficiently reach affected cells11.
The formation of liposome surface complexes with the
previously mentioned functionalized chitosan makes it feasible to combine
various delivery methods. Therefore, a combination of liposomal delivery
systems for anti-tuberculosis medications and complexes of antibacterial
medicines with derivatives of β-cyclodextrin, bound together by a
mucoadhesive polymer, might greatly improve treatment efficacy and shorten
treatment duration.
Anti-tuberculosis medications are delivered by a
variety of combined systems. For instance, compared to alginate-modified PLGA
nanoparticles containing a single medication, a synergistic effect of the
double capture of moxifloxacin and amikacin was demonstrated in12.
As evidenced by the determination of moxifloxacin and prednisolone using
reverse-phase high-performance liquid chromatography in13, the
development of such systems necessitates the development of techniques for the
simultaneous registration of multiple drug molecules in pharmaceutical
preparations.
Instrumentation:
The separation was carried out on HPLC System with
Waters 2695 alliance with binary HPLC Pump, Waters 2998 PDA detector, Waters
Empower2 Software with Agilent Zorbax Eclipse XBD-C8, (150mm×4.6;5µm) column.14
2. MATERIALS AND METHODS:
2.1 Materials:
Mono-(6-(hexamethylenediamine)-6-deoxy)-β-cyclodextrin
(NH2-CD), 2-hydroxyPropyl-β-cyclodextrin (HP-CD), 5 kDa Chitosan
oligosaccharide lactate with deacetylation Degree 98% (Chit), levofloxacin and
rifampicin were obtained from Sigma Aldrich (St. Louis, USA); tablets of
phosphate-buffered saline and HCl were obtained from “Pan Eco”(Moscow, Russia);
dipalmitoyl phosphatidylcholine, sodium salt and 16:0 cardiolipin
10,30-Bis-[1,2-dipalmitoyl-sn -glycero-3-phospho]-glycerol were obtained
Mono-(6-(hexamethylenediamine)-6-deoxy)-β-cyclodextrin (NH2-CD),
2-hydroxypropyl-β-cyclodextrin (HP-CD), 5 kDa Chitosan oligosaccharide
lactate with deacetylation degree 98% (Chit), levofloxacin and rifampicin were
obtained from Sigma Aldrich (St. Louis, MO, USA); tablets of phosphate-buffered
saline and HCl were obtained from “Pan Eco”(Moscow, Russia); dipalmitoyl
phosphatidylcholine, sodium salt and 16:0 cardiolipin
10,30-bis-[1,2-dipalmitoyl-sn -glycero-3-phospho]-glycerol were obtained from
“Avanti Polar Lipids” (Alabaster, AL, USA); dialysis bags with a cut-off
molecular weight of 12–14 kDa were obtained from “Orange Scientific”
(Braine-ladled, Belgium); and dialysis bags with a cut-off molecular weight of
3,5 kDa were obtained from “Serva” (Heidelberg, Ger-many). NH2-CD-Chit and
HP-CD-Chit were synthetized and purified according to the methodic and used in
this research without additional treatment. From “Avanti Polar Lipids”
(Alabaster, AL, USA); dialysis bags with a cut-off molecular weight of 12–14
kDa Were obtained from “Orange Scientific” (Braine-ladled, Belgium); and
dialysis bags with a cut-off molecular weight of 3,5 kDa were obtained from
“Serva” (Heidelberg, Ger-Many). NH2-CD-Chit and HP-CD-Chit were synthetized and
purified according to the Methodic15 and used in this research
without additional treatment.
2.2. Liposomal form of Rif Preparation:
Liposomes were obtained through lipid film hydration
followed by sonication. Solutions of DPPC and CL in chloroform (25 mg/mL) in
the required mass ratio (DPPC 100 w. % and DPPC:CL w. % 80:20) and were placed
in a round bottom flask, then the Solvent was removed on a rotary evaporator at
a temperature below 55 ◦C. The resulting thin film was dispersed in 0.01M
sodium phosphate-buffered solution (pH = 7.4) to a lipid Concentration of 5mg/mL,
then the flask was exposed to an ultrasonic bath (37 Hz) for 5 min. The opaque
suspension was transferred into a plastic tube and sonicated (22kHz) For 10min
continuously with constant cooling on a 4710 Cole-Parmer Instrument disperser.
Liposomal forms of rifampicin were obtained in a similar way with some changes:
A thin lipid film was dispersed in 0.01M sodium phosphate-buffered solution (pH
= 7.4) Containing rifampicin at a concentration of 2 mg/mL. The unloaded drug
was separated by dialysis against 0.01M sodium phosphate-buffered solution (pH
= 7.4) in Serva dialysis bags with a cut-off molecular weight of 3500 Da for
120 min at 4◦C.
The encapsulation efficiency (EE) of rifampicin in
liposomes was calculated according to Equation (1):
v(Rif)total –
v(Rif)dialysis
EE = ––––––––––––––––––––––––
× 100%
v(Rif)
total
where ν(Rif)total is the total amount of Rif in
the initial system before dialysis and ν(Rif)dialysis is the amount of Rif
determined in the external solution after dialysis against sodium phosphate-buffered
solution 0.01M for 120 min at a temperature of 4◦C. Complexes
of liposomes with conjugates of chitosan and β-cyclodextrin were obtained
By adding a solution of NH2-CD-Chit or HP-CD-Chit (5mg/mL) (loaded with Lev or
Empty) to a solution of liposomes (5mg/mL) in a sodium phosphate-buffered
solution (pH = 7.4) at a base-molar ratio of 7:1. The complexes were incubated
at room temperature For 15 min.
2.3. Complexes of Levofloxacin with the Conjugate of
Chitosan and B-Cyclodextrin Preparation.
The solution of levofloxacin in hydrochloric acid (pH
4.0, 3mg/mL) was combined with a solution of NH2-CD-Chit (5mg/mL) with the same
pH at the same ratio to achieve 2×excess of CD-tori in relation to Lev
molecules. The complexes were incubated at 37◦C For 60min.
The encapsulation efficiency (EE) of Lev in carrier
was calculated according to Equation (2):
N(Lev)total
– v(Lev)dialysis
EE
= ––––––––––––––––––––––––– ×100%
v(Lve)total
Where ν(Lev)total is the total amount
of Lev in the initial system before dialysis and Ν(Lev)dialysis
is the amount of Lev determined in the external solution after dialysis against
A 1·10−4 M HCl solution in Serva dialysis bags with a cut-off molecular
weight of 3500 Da For 15min at a temperature of 4◦C.
UV-Visible Spectroscopy:
UV-visible spectroscopy relies on energy, radiation,
or electron excitation. The UV-Visible technique uses energy light to excite
electrons. The sample wavelength is determined by the absorbance range of 200
to 800nm. Absorption occurred only when conjugated pi-electrons were present.16
FTIR Spectroscopy:
Infrared spectroscopy shifts absorption to a lower
energy state, resulting in vibration and excitation of atoms and molecules.
This approach identifies the functional group and original peaks of a molecule,
allowing scientists to design new methods.17
Mass Spectroscopy (MS):
Mass spectroscopy involves ionizing molecules with
high-energy electrons. The mass of each charge was carefully detected and
evaluated using magnetic field fluctuations and electrostatic wave
acceleration, ensuring correct weight of molecules.18
Nuclear Magnetic Resonance Spectroscopy (NMR):
Scientists have developed many approaches to analyze
novel medication compounds. Nuclear magnetic resonance spectroscopy was
commonly employed in medication development.15 This technique proved
effective in identifying and analyzing medications using quantitative analysis
to determine their molecules. This approach is useful for identifying drug
composition, chemical products, pharmaceutical formulations, and biological fluids.19
Chromatographic Technique:
High Performance Thin Layer Chromatography (HPTLC):
This approach is widely used to identify, estimate,
and assess the analytical profile of pharmacological compounds. This advanced
technology will play a significant role in drug analysis.17 Its quick
separation action and flexibility make it suitable for analyzing various
medication components in the pharmaceutical industry. This technique has the
advantage of allowing for quick drug analysis, easy handling, and cleaning of
crude drug samples. This technique allows us to characterize chromatograms
without time constraints for several parameters.20
High Performance Liquid Chromatography (HPLC):
High-performance liquid chromatography is a key
technology for separating complicated mixtures of chemicals and molecules. This
approach effectively targets chemical substances and biological components.19
In 1980, this approach was invented. With the adoption of HPLC, it became the
first method to analyze bulk drug compounds as per USP-1980 standards. To
ensure accuracy, precision, and a diverse range of samples, the HPLC method was
used prior to drug analysis. A UV detector was utilized to estimate samples by
HPLC and determine their wavelength. The UV detector procedure begins after
numerous wavelength scanning programs have been applied.21
Thin Layer Chromatography (TLC):
Thin layer chromatography is a traditional method for
analyzing chemicals in pharmaceuticals. This approach uses two phases: the
mobile phase and the stationary phase.21 To prepare samples, the
solid phase, adsorbent, and a thin layer of silica gel were distributed on a
glass plate with an aluminum support. This approach was frequently used to
analyze both inorganic and organic substances was chosen for compound analysis
due to its minimal cleaning requirements, flexible mobile phase selection, high
sample loading capacity, and cost effectiveness. This approach proved
particularly useful for analyzing bulk medication components.22
Gas Chromatography:
This technique effectively separates volatile and
organic components. Gas chromatography separates compounds for quantitative
measurement of numerous medication combinations, including compound tracing and
parts per trillion. Gas chromatography is essential for analyzing
pharmaceutical drugs and identifying contaminants.23
HPLC Conditions:
The mobile phase consisting of water (pH Adjusted with
Ortho phosphoric acid: Methanol (HPLC grade) were filtered through 0.45µ
membrane Filter before use, degassed and were pumped from the solvent reservoir
in the ratio of 80:20v/v was Pumped into the column at a flow rate of
0.5ml/min. The column temperature was 40°C. The detection Was monitored at
270nm and the run time was 6min. The volume of injection loop was 10µl prior To
injection of the drug solution the column was Equilibrated for at least 15min. with
the mobile phase Flowing through the system.
Preparation of standard solution:
Ten milligrams of standard RIF and lev were accu Ratley
weighed, transferred to 10ml volumetric flasks Separately, dissolved in
acetonitrile and then volumes Were made up to the mark with acetonitrile, to obtain
Solution containing 1000µg/ml. Aliquots of the stock Solutions were
appropriately diluted with acetonitrile to Obtain working standards of 100µg/ml
solutions of RIF And lev.
2.4. Chromatographic conditions:
Various solvents in different ratios such as water and
Acetonitrile along with potassium dihydrogen Phos-Phate buffer were tried. The
chromatographic separa-Tion was achieved on Kinetics C18, 100 A Phenomenex
Column (250mm × 4.6mm, 5µm) at room temperature (25±2°C) using optimized mobile
phase consisting of 0.03M potassium dihydrogen phosphate buffer pH 3.0:
Acetonitrile (55:45). The mobile phase flow rate was 0.8 ml/min, injection
volume 10µL and run time was 10 min. The analysis was performed at 230nm, using
Mobile phase as diluent. The standard solutions of different concentrations
were filled in vials and kept in Autosampler. The mobile phase was prepared
daily, degassed by ultrasonicate and filtered through a 0.45-µm membrane filter
prior to use.
2.5. Method validation:
The ICH Q2 (R1) guideline and the USP system
suitability test were used to assess the HPLC method’s linearity, sensitivity,
precision, accuracy, and robustness24,25.
2.5.1. System suitability test:
Five replicate injections of 1 and 2µg/ml standard
solutions of RIF and lev, respectively, were used in the system suitability
test. Peak area, tailing factor, theoretical plates, resolution, retention
duration, and other factors were assessed.
2.5.2. Linearity:
An analytical method’s linearity is its capacity to
yield results that are directly, or via a mathematical transformation,
proportionate to the analyte’s concentration within a certain range. In five
replicates, varying amounts of standard drug solutions were injected to achieve
a concentration range of 1–5µg/ml for RIF and 2–10µg/ml for lev. Ordinary
linear regression analysis was used to evaluate the linearity in terms of
recorded peak areas vs matching medication concentrations. The correlation
coefficient (r2), slope, and intercept (with corresponding confidence ranges)
were computed and assessed. Additionally, the Bartlett’s test was used to
confirm the homoscedasticity of the variances along each drug’s regression line26.
2.5.3. Sensitivity:
The sensitivity of method was measured in terms of
Limit of Detection (LOD) and Limit of Quantification (LOQ). LOD and LOQ of the
developed method were calculated from the standard deviation of the response
and Slope of the calibration curve of drugs using the formula as per ICH
guideline,
Limit of detection = 3.3 × σ/S
Limit of quantitation = 10 × σ/S
Where, “σ” is standard deviation of y intercepts
of Regression lines, “S” is Slope of calibration curve.
2.5.4. Precision:
Intra-day and inter-day precision studies were used to
assess the developed method’s accuracy. Three repetitions of three different
concentrations (1, 3, and 5 µg/ml for RIF; 2, 6, and 10µg/ml for lev) were
performed on the same day in order to achieve intra-day precision. The peak
area observed was represented in terms of percent relative standard (% RSD).
Using the specified concentrations of both medications in duplicate, the
inter-day precision research was carried out on three separate days, and the
percentage RSD was computed.
2.5.5. Accuracy:
The standard addition method27, which
involves spiking a sample containing a synthetic mixture of rifampicin,
levofloxacin, and PLGA at three distinct concentration levels of 50%, 100%, and
150%, was used to evaluate the method’s accuracy. In summary, recovery tests
were conducted by adding three different concentrations of lev standard (2, 4,
and 6µg) and RIF standard (1, 2, and 3 µg) to the synthetic mixture that
contained lev (4µg/ml) and RIF (2µg/ml). Recovery trials were carried out in
triplicate using the recovery and percentage RSD for each medication.
2.5.6. Robustness testing using 24–1 fractional
Factorial design:
The current study used a 24–1 fractional factorial
design (FFD) to assess the robustness of the HPLC analytical approach for the
simultaneous quantification of rifampicin (RIF) and levofloxacin (lev) Four
independent parameters were chosen for the current investigation based on
chromatographic intuition, experience from earlier research, and the
criticality of components seen during trial runs. The impact of variables such
as acetonitrile volume in mobile phase composition (A), buffer concentration
(B), flow rate (C), and wavelength (D) on the drug retention time, resolution,
and asymmetry factor of lev was investigated.
The ranges of components tested were purposefully
altered from the optimal technique settings of both medications in order to
objectively analyze the departure of the considered answers from the original
value. Table 1 displays the four factors along with their intentional
variations in terms of high and low levels. Design Expert (Version 10.0,
Stat-Ease Inc., Minneapolis, MN, USA) statistical software was used to analyze
the generated data. To reduce the bias effects of uncontrollable factors, every
experiment was conducted in a randomized order.
2.6. Analysis of synthetic mixture:
The synthetic drug mixture of RIF and lev with PLGA
75:25 polymer was made, and the assay of the medications in the synthetic
mixture was examined using the method described below. In order to create a dry
powder inhaler comprising the two medications with sustained release qualities,
PLGA 75:25 was selected as the polymer for the synthetic combination. RIF: lev
(1:2) and drugs: polymer (1:1) made up the synthetic mixture, which mimicked
the makeup of the formulation.
Table 1: Experimental factors and levels used in FFD.
|
Factors
|
High level
|
Low level
|
|
A-Acetonitrile
volume in mobile phase Composition (ml)
|
50
|
40
|
|
B-Buffer
Concentration (M)
|
0.03
|
0.02
|
|
C-Flow rate
(ml/min)
|
1.0
|
0.8
|
|
D-Wavelength (nm)
|
232
|
228
|
A 10ml volumetric flask was filled with precisely
weighed 60mg of synthetic combination equivalent to 10mg of RIF and 20mg lev,
which were then dissolved in 5 ml of acetonitrile. The solution was then
sonicated for five minutes, and acetonitrile was added to bring the volume up
to ten milliliters. After filtering the mixture through Whatman filter paper
number 42 that had been wetted with acetonitrile, the mixture was further
diluted to yield 1 µg/ml of RIF and 2µg/ml of lev.
3. RESULTS AND DISCUSSION:
3.1. Optimization of chromatographic conditions:
The chromatographic conditions were optimized. Carried
out in order to create an HPLC technique for the simultaneous measurement of
lev and RIF in bulk and in the form of a pharmacological dosage. Regarding the
choice of wavelength, 10µg/ml RIF standard solutions, and lev were examined in
spectral mode between 200 and 400nm with a blank of acetonitrile. Both
medications significantly absorbed at 230nm, which was used as the wavelength
of detection.
Various mobile phases comprising different ratios of
water, acetonitrile and potassium dihydrogen Phos-Phate buffer were tried.
Water and acetonitrile when used as mobile phase in different ratios lead to
early Elution of both drugs giving sharp peak of RIF but tail-Ing was observed
in lev peak. Hence, various ratios of different strengths of potassium
dihydrogen Phos-Phate buffer and acetonitrile were tried that gave Acceptable
peak shape of RIF and lev and resolution. Finally, mobile phase comprised of 0.03M
potassium Dihydrogen phosphate adjusted to pH 3.0 with ortho- Phosphoric acid
and acetonitrile in ratio of 55:45 v/v Gave acceptable retention time lev
(2.91±0.447 min) And RIF (4.87 ± 0.395min) at 230nm and 0.8ml/min Flow rate.
The injection volume to carry out chromato-Raphy was set at 10µL.
3.2. System suitability parameters:
System suitability testing in HPLC method showed That
the method was suitably performed under the Optimized conditions, and %RSD was
found less Than 2%, for system suitability parameters: Rt (For RIF, 4.87±0.395;
for lev, 2.91±0.448), area (For RIF,23714.2±1.083; for lev, 76786.4±0.122), and
res-Olation (7.42±0.343). Moreover, theoretical plates, 3576.34±0.893 and
7262.54 ±1.819 as well as tailing Factor 1.35±0.523 and 1.266± 0.706 for lev
and irrespectively were obtained.
3.3. Method validation:
3.3.1. Linearity:
An analytical method’s linearity is defined as its
capacity to yield results that are directly, or via a mathematical
transformation, proportionate to the analyte’s concentration within a certain
range. In the suggested concentration range of 1–5µg/ml for RIF and 2–10 µg/ml
for lev, the RIF and lev demonstrated a strong correlation coefficient (r 2 =
0.9985 and 0.9994, respectively). Bartlett’s test verified the homoscedasticity
of variance, and the peak area response for both medications demonstrated
homogeneous variance, as demonstrated by the χ2 value being lower than the
tabulated value (Table 2). As a result, no additional weighting or
transformation method was required based on the results. The overlay HPLC
chromatogram for RIF and lev linearity at 230nm.
3.3.2. Sensitivity:
LOD for RIF and lev was found to be 0.0921 and 0.0914
µg/ml while LOQ was found to be 0.2790 and 0.2771 µg/ml respectively indicating
the sensitivity of Method (Table 2).
Table 2: Analytical validation parameters for RIF and
lev by HPLC method.
|
Parameters
|
RIF
|
Lev
|
|
Linearity
|
|
Linearity range
(μg/ml)
|
1–5
|
2–10
|
|
Correlation
coefficient (r2)
|
0.9985
|
0.9994
|
|
Slope ± SD
|
25474.58 ± 275.03
|
30423.40 ± 152.64
|
|
Confidence limit
of slope
|
25133.09–25816.07
|
30233.87–30612.93
|
|
Intercept ± SD
|
1373.66 ± 71.08
|
10661.50 ± 842.92
|
|
Confidence limit
of intercept
|
1285.52–1461.80
|
9614.87–11708.12
|
|
Bartlett’s test
(χ2)
|
0.0031
|
0.0007
|
|
Sensitivity
|
|
LOD (μg/ml)
|
0.0921
|
0.0914
|
|
LOQ (μg/ml)
|
0.2790
|
0.2771
|
|
Precision(%RSD)
|
|
Intra-day
Precision
|
0.163–0.811
|
0.098–0.366
|
|
Inter-day
Precision
|
0.449–1.818
|
0.229–0.373
|
|
Accuracy
|
|
50%
|
100.50 ± 0.05
|
98.14 ± 0.034
|
|
100%
|
99.37 ± 0.08
|
101.32 ± 0.077
|
|
150%
|
100.80 ± 0.17
|
99.29 ± 0.077
|
SD = standard deviation, % RSD = relative standard
deviation,
a) RIF = Rifampicin, lev = levofloxacin an Average of
five determinations.
b) Confidence interval at 95% confidence level and 5
degrees of freedom (t = 2.57).
c) Calculated value less than tabulated value, χ2
critical value 9.488 at α = 0.05.
d) Average of three determinations for each
concentration.
e) Average of three determinations at each level.
3.3.3. Precision:
The experiment was repeated three times in a day
(Intraday precision) and the average %RSD values of the results were
calculated. Similarly, the experiment was repeated on three different days
(Inter-day precision) and the average %RSD values for peak area of RIF and OFX
was calculated. Results of intra-day and inter day precision expressed in terms
of %RSD less than 2 confirm precision of the method (Table 2)
3.3.4. Accuracy:
After
spiking with standard, the mean percentage recovery at three levels—50%, 100%,
and 150%—was between 99.37 and 100.80% for RIF and 98.14 and 101.32% for lev,
all of which fell within acceptable ranges of 100±2% (Table 2). The method’s
accuracy was confirmed by finding good agreements between determined and actual
values. The method’s appropriateness and applicability for regular drug
analysis are suggested by the percentage RSD less than 2 for both substances.
3.3.5. Robustness:
According to the ICH, an analytical procedure’s
robustness is its ability to withstand minor and intentional changes in
methodology28. Four independent factors—acetonitrile volume in
mobile phase composition (A), buffer concentration (B), flow rate (C), and
wave-length (D)—were chosen for this study based on trial runs’ criticality,
chromatographic intuition, and prior optimization study experience. The
retention factor of RIF (Response 1), retention factor of lev (Response 2),
resolution of lev (Response 3), and asymmetry factor of lev (Response 4) were
the qualitative responses examined in the tests, which were conducted according
to the experimental domain. The correlation of the factors’ effects on each
drug’s retention factor was displayed graphically using response surfaces and
perturbation plots. The steepest slope or curvature demonstrates sensitivity to
particular circumstances, and perturbation plots show the change in response
from its nominal value with all other parameters held constant at a reference
point. Effects above the Bonferroni Limit are almost certainly significant,
effects above the t-value limit are perhaps significant, and effects below the
t-value limit are not likely to be significant. The Pareto chart is helpful for
determining the significance of factors. The Pareto chart shows that, in
decreasing order, each choice had a significant impact on the chosen responses:
A > C > B > AB > D for response 1; for response 2, C > A > B
= AB > D = AC = AD; for Response 3, A > C > B > AD > AC > D;
and for Response 4, C > AB > A = D > B, as illustrated in Figure 4. As
seen in Figure 5, perturbation plots revealed that even slight changes in
acetonitrile volume and flow rate had a substantial impact on each response.
The third variable was kept constant at a predetermined level, typically the
suggested optimum, while the equation-based 3D response surface plots were
produced as a function of the important factors. The three-dimensional response
surface plots show that an increase in the volume of acetonitrile content of
the mobile phase and flow rate resulted in a decrease in the resolution of both
medicines and an increase in Rt of RIF, Rt of lev, and as of lev.
Using Design Expert software, analysis of variance
(ANOVA) was used to validate the model (Table 3). Predictions about the
reaction for specific amounts of each element can be made using the equation in
terms of coded or actual factors. By comparing the factor coefficients, the
coded equation can be used to determine the relative impact of the components.
A robust approach is produced when the model’s p value is greater than 0.05,
which indicates that the components had no significant impact on the response.
A strong correlation between the experimental data and the fitted models is
shown by the low standard deviation [% coefficient of variance (CV)] and
sufficient precision. In a polynomial equation, a positive sign denotes a
synergistic impact, whereas a negative sign denotes an antagonistic effect. In
summary, among the four variables, the volume of acetonitrile in the mobile
phase and flow rate seemed to have a potentially considerable impact on the
asymmetry factor of levofloxacin and the retention factor of both medicines,
therefore it was crucial to be carefully regulated.
3.4. Analysis of synthetic mixture:
Using the suggested HPLC procedure in triplicate, the
concentration of RIF and lev in the synthetic mixture was examined. The
suggested approach may be successfully used to the examination of formulation
including RIF and lev, and the percent assay discovered within the range of
100.258–103.19% with %RSD less than 2 indicates the absence of interference
from PLGA 75:25.
4. CONCLUSION:
It made sense to create a quick and easy HPLC approach
for the simultaneous measurement of ofloxacin and rifampicin. The makeup of the
mobile phase The settings that produced the best peak parameters were used to
optimize the chromatographic conditions. The suggested approach was thoroughly
validated and found to be robust, accurate, linear in the concentration range
under study, sensitive, and precise in identifying RIF and OFX in a synthetic
mixture. LOQ values for both substances verified the method's capacity to
quantify a little number of drugs. The simultaneous variation of effects on
responses was studied using FFDonrobustness. Considering the With the exception
of the acetonitrile content in the mobile phase and flow rate, responses,
buffer concentration, and wavelength were all strong. The only important
factors influencing robustness studies were acetonitrile's mobile phase and
flow rate, which needed to be regulated. It is determined that using
experimental design and response surface approach is a flexible process that
can lower the number of experiments required for the robustness analysis of the
HPLC method. The suggested HPLC approach would be useful for combined dose form
analysis and routine quality control.
5. REFERENCES:
1.
World Health
Organization. WHO global tuberculosis Report. Geneva: WHO; 2016.
2.
Hall RG, Leff RD, Gumbo
T. Treatment of active pulMonary tuberculosis in adults: current standards and
Recent advances. Pharmacother. 2009; 29: 1468–1481.
3.
O’Neil MJ, editor. The
merck index an encyclopedia of chemicals, drugs, and biologicals. 13th ed.
Whitehouse Station (NJ): Merck and Co., Inc.; 2001. p. 1474.
4.
Maggi N, Pasqualucci CR,
Ballota R, et al. Rifampicin: A new orally active rifamycin. Chemother. 1966; 11:
285–292.
5.
Rees RJW, Pearson JMH,
Waters MFR. Experimental and Clinical studies on rifampicin in treatment of
leprosy. Br Med J. 1970; 1: 89–92.
6.
Binda G, Domenichini E,
Gottardi A. Rifampicin, a general Review. Arzneim Forsch. 1971; 21: 1907–1977.
7.
Pähkla R, Lambert J,
Ansko P, et al. Comparative bioavail-Ability of three different preparations of
rifampicin. J Clin Pharm Ther. 1999; 24: 219–225.
8.
Tsankov N, Angelova I.
Rifampin in dermatology. Clin Dermatol. 2003; 21: 50–55.
9.
Berning, S.E. The role
of fluoroquinolones in tuberculosis today. Drugs 2001; 61: 9–18.
10.
Jacobs, M.R. Activity of
quinolones against mycobacteria. Drugs 1999; 58: 19– 22. [CrossRef]
11.
Pham, D.D.; Fattal, E.; Tsapis,
N. Pulmonary drug delivery systems for tuberculosis treatment. Int. J. Pharm. 2015;
478: 517–529.[CrossRef]
12.
Chopra, H.; Mohanta, Y.K.;
Rauta, P.R.; Ahmed, R.; Mahanta, S.; Mishra, P.K.; Panda, P.; Rabaan, A.A.; Alshehri,
A.A.; Othman, B.; et al. An insight into advances in developing nanotechnology based
therapeutics, drug delivery, diagnostics and vaccines: Multidimensional applications
in tuberculosis disease management. Pharmaceuticals 2023; 16: 581. [CrossRef]
13.
Razzaq, S.N.; Khan, I.U.;
Mariam, I.; Razzaq, S.S. Stability indicating HPLC method for the simultaneous determination
of moxifloxacin and prednisolone in pharmaceutical formulations. Chem. Cent. J.
2012; 6: 94. [CrossRef]
14.
http://dx.doi.org/10.13005/msri/110109(Received:
August 15, 2014; Accepted: August 29, 2014)
15.
Le-Deygen, I.M.; Skuredina,
A.A.; Mamaeva, P.V.; Kolmogorov, I.M.; Kudryashova, E.V. Conjugates of chitosan
with β-Cyclodextrins as promising carriers for the delivery of levofloxacin:
Spectral and microbiological studies. Life 2023; 13: 272.[CrossRef]
16.
Vishwanathan K, Bartlett
MG, Stewart JT. Determination of Gatifloxacin in human plasma by liquid Chromatography/
electrospray tandem mass spectrometry. Rapid Communications in Mass Spectrometry.
2001; 15(12): 915-9.
17.
Singh RK, Rathnam MV, Singh
SJ, Vegesna RV. Determination of Camylofin dihydrochloride and Nimesulide in Pharmaceutical
Preparation by Gas chromatography. American Journal of Analytical Chemistry. 2011;
2(8): 944.
18.
Natesan S, Thanasekaran D,
Krishnaswami V, Ponnusamy C. Improved RP-HPLC method for the simultaneous estimation
of Tranexamic acid and mefenamic acid in tablet dosage form. Pharm. Anal. Acta.
2011; 2(1): 115.
19.
Puozzo C, Filaquier C, Zorza
G. Determination of milnacipran, a Serotonin and noradrenaline reuptake inhibitor,
in human Plasma using liquid chromatography with spectrofluorimetric Detection.
Journal of Chromatography B. 2004; 806(2): 221-8.
20.
Shinozuka T, Terada M, Tanaka
E. Solid-phase extraction and Analysis of 20 antidepressant drugs in human plasma
by LC/MS With SSI method. Forensic Science International. 2006; 162(1-3): 108-12.
21.
Zhang LJ, Yao YM, Sun JJ,
Chen J, Jia XF. An LC–MS/MS Method for Simultaneous Quantification of Seven Anti-HIV
Medicines in Plasma of HIV-infected Patients. Pharm Anal Acta. 2010; 1(1): 1.
22.
Rajender G, Narayana NG.
Liquid Chromatography-Tandem Mass Spectrometry Method for Determination of Paclitaxel
in Human Plasma. Pharm Anal Acta. 2010; 1: 101.
23.
Sharma HK, Jain N, Jain SK.
Development of spectrophotometric Method for quantitative estimation of Amlodipine
besylate, Olmesartan medoxomil and hydrochlorthiazide in tablet dosage Form. Pharm
Anal Acta. 2011; 2(126): 2.
24.
International Conference
on Harmonization. ICH Q2 (R1): validation of analytical procedures: text and methodology,
ICH Secretariat, Geneva, 2005.
25.
United States Pharmacopeia
27, national formulary 22,vol.2, The United States Pharmacopoeial Convention, Rockville,
2003. p. 2281.
26.
Zar JH. Biostatistical analysis.
5th ed Upper saddle River(NJ): Pearson Educational Publications; 2010.
27.
Kleinschmidt G. Method validation
in pharmaceutical analysis. In: Ermer J, Miller JHM, editor. A guide to best practice.
Weinheim: Wiley-VCH Verlag GmbH and Co. KGaA; 2005. p.70
28.
Annapurna MM, Mohapatra C,
Narendra A. Stability-indicating liquid chromatographic method for the deter-mination
of Letrozole in pharmaceutical formulations. J Pharm Anal. 2012; 2: 298–305.
