Determination of Flucloxacillin
Sodium by Analytical Spectrophotometry
Saad Antakli, Leon Nejem, Walaa Alsheikh Ahmad
Department of Chemistry, Faculty of Science, University
of Aleppo, Syria.
*Corresponding Author E-mail: antakli@scs-net.org
ABSTRACT:
Simple
and rapid spectrophotometric method has been developed for the determination of
Flucloxacillin sodium (FLU) in raw material. The method is based on the formation
of yellow ion-pair complex between Flucloxacillin sodium and Bromocresol purple
(BCP) in Chloroform medium.
The absorption maximum of the complex was found to be 408 nm. Different parameters
affecting the reaction were optimized such as: effect of solvents, time,
reagent concentration, correlation ratio, etc. The formed complex was quantified
spectrophotometrically at absorption maximum. Linearity range was 2.47 – 22.23 µg/mL,
regression analysis showed a good correlation coefficient R2 = 0.9999.
The limit of detection (LOD) and limit of quantification (LOQ) were to be 0.203
µg/mL and 0.615 µg/mL. The method was successfully applied for the determination
of Flucloxacillin sodium in raw material. The proposed method is simple, direct,
sensitive and do not require any extraction process. Thus, this method could be
readily applicable for the quality control and routine analysis.
KEYWORDS: Flucloxacillin sodium, Bromocresol purple, Spectrophotometric method.
INTRODUCTION:
Flucloxacillin sodium is a semisynthetic
penicillin’s, chemically known as; (6R)-6-[3-(2-chloro-6-fluorophenyl)-5-methylisoxazole-4-carboxamido]
penicillanic sodium Fig. (1, a). It contains a large phenyl-substituted
isoxazolyl moiety which protects the β-lactam bond (N1-C7) by steric hindrance
from hydrolysis by a variety of β-lactamases, including cephalosporinases,
penicillinases, and extended spectrum beta-lactamases. It is used primarily to treat
infections caused by bacteria that are resistant to other penicillin-type antibiotics,
but when Flucloxacillin-sodium prescribed on its own, it is less effective than
more commonly used penicillin-type antibiotics, so it is often prescribed in combination
with other penicillins to ensure an extended spectrum of efficacy in treatment of
joint infections, pneumonia, and toxic shock syndrome1. Several methods have been reported in
the literature for the analysis of Flucloxacillin sodium such as Thin-layer chromatography
(TLC)2, Liquid chromatography (LC)3, Spectrophotometric method4-5-6-7-8, Reverse phase high performance liquid
chromatographic (RP-HPLC) method9-10-11.
Bromocresol purple
(BCP) as shown in Fig. (1, b) is a sulphonphthalein dye commonly used as indicator
and spectrophotometric reagent.
Fig.
(1, a): Structural formula of Flucloxacillin
sodium.
Fig. (1, b):
Structural formula of Bromocresol purple.
MATERIALS
AND METHODS:
Apparatus:
All
spectral measurements were carried out using a Spectro Scan 80 DV, UV/Vis spectrophotometer instrument Ltd
(UK), connected to computer, quartz cells 1 cm. Ultrasonic bath Daihan (China), and stirrer Velp Scientifica (Europe).
Chemical
regents:
Chloroform from Merck (Germany) was used to prepare
the Flucloxacillin sodium solutions.
Methanol from Lobal Chemie (India) was used to dissolve the standard. Flucloxacillin
sodium standard material
purity 99.3%, was obtained
from China. Bromocresol purple from Merck (Germany).
Standard
preparation:
Flucloxacillin
sodium stock solutions:
Stock
solutions 5×10-3 M of Flucloxacillin
sodium (MW = 493.9
g/mol) was prepared by dissolving 24.70 mg of Flucloxacillin sodium standard material equivalent to 24.87 mg (by taken the
purity in consideration) in volumetric flask 10 mL of Methanol, then 1 mL of the
solution was taken to volumetric flask 10 mL and diluted with Chloroform to give concentration 5×10-4 M equivalent to 246.95
𝜇g/mL. The
working standard solutions of Flucloxacillin
sodium standard material samples were prepared by appropriate dilutions
among (50 - 1000) 𝜇L by suitable dilution of Flucloxacillin sodium 246.95 𝜇g/mL in several volumetric flasks 10 mL and added for each
1 mL of Bromocresol purple 5×10-3 M, then completed to volume with Chloroform to give concentrations
between (1.23- 24.70) 𝜇g/mL of Flucloxacillin
sodium.
Reagent stock solution:
Bromocresol purple
5×10-3 M was prepared by dissolving 135.055 mg of Bromocresol purple
(MW = 540.22 g/mol) in volumetric flask 50
mL and completing to volume with Chloroform.
Calibration
curve:
To
construct the calibration curve, five standard Flucloxacillin sodium solutions for each
concentration were prepared and the
absorbance was measured of each solution five times.
RESULTS AND DISCUSSION:
Flucloxacillin sodium forms with Bromocresol purple at 25 ± 5şC yellow ion-pair complex and stability of the complex was 10 hours. The result solution was scanned in the range of wavelengths 300-550 nm against a blank of BCP prepared in Chloroform, and then measured the absorbance at maximum wavelength 408 nm. We studied all the parameters of the colored result solutions to obtain the optimal conditions. Fig. 2 shows the spectrum of complex between Flucloxacillin sodium and Bromocresol purple in Chloroform medium.
Fig. 2: a- Spectrum of complex FLU-BCP in Chloroform medium (1.5×10-5 M).
b- Spectrum of BCP in Chloroform medium (1.5×10-4 M).
Stability of stock solution:
Time effect on the stability of standard stock solution of Flucloxacillin sodium in Methanol was studied in three different concentrations 2.5×10-4 M, 5.0×10-4 M and 7.5×10-4 M. We did not notice any significant changes during the absorption measurement within two months. Fig. 3 presented the stock solution stability of Flucloxacillin sodium in Methanol during the working period.
Fig. 3: Effect of time on stock standard solutions
of Flucloxacillin sodium in
Methanol.
Stability of formed complex:
Time effect on the stability of complex between Flucloxacillin sodium and Bromocresol purple in Chloroform medium was studied in three different concentrations 9.88, 14.82 and 19.76 µg/mL. We did not notice any significant changes during the absorption measurement within 10 hours. Fig. 4 presented the solution stability of complex between Flucloxacillin sodium and Bromocresol purple in Chloroform medium during the working period.
Fig. 4: Effect of time on of complex between Flucloxacillin
sodium and Bromocresol purple in Chloroform medium.
Effect of reagent concentration:
To study the effect of reagent concentration on the colored
complex solution, we made a series of 10 mL of separated volumetric flasks, by adding
0.9 mL of Flucloxacillin sodium
stock solution to obtain 5×10-4
M equivalent to 45 µM
and added between (0.04 - 1) mL of (BCP) 5×10-3 M, equivalent to (20 -
500) µM, and completed to 10 mL by Chloroform. The absorbance at 408 nm for every added (BCP) reagent was measured
against the blank of Chloroform. It was found that the completed colored complex formation was 0.9 mL of
(BCP) solution as it is shown in Fig. 5. The best concentration addition of (BCP)
was 450 µM of Flucloxacillin sodium
(CBCP = 10 CFLU).
Fig. 5: Effect of reagent concentration.
Correlation ratios by molecular ratio:
We have prepared a series of complex solutions FLU-BCP in the medium of the Chloroform. The concentration of the reagent changes within the ratio (5×10-6 - 1×10-4) M while the concentration of Flucloxacillin sodium was constant in each solution and equal to 2.5×10-5 M.
We measured the absorbance values of these solutions at the wavelength of the maximum absorbance 408 nm according to the used reagent percentage (using Chloroform as a blank). The absorption changes of the molecular ratio of the reagent to the Flucloxacillin sodium permitted to measure correlation ratio, we obtained the curve A = f ([BCP]/[FLU]) shown in Fig. 6 where the correlation ratios are (1:1 & 2:1).
Fig. 6: Correlation molecular ratios (1:1 & 2:1).
Correlation ratios by continuous variation:
We have prepared a series of complex solutions FLU-BCP in the medium of the Chloroform. The concentration of the reagent and the concentration of Flucloxacillin sodium changes in solutions between (0 - 1×10-4 M) where the sum of both concentrations remains constant and equal to 1×10-4 M.
We measured the absorbance values of these solutions at the wavelength of the maximum absorbance 408 nm according to the used reagent percentage of the formed complex in terms of molecular fraction of Flucloxacillin sodium. we obtained the curve A = f ([BCP] /{[BCP] + [FLU]}) shown in fig. 7: where the correlation ratios are also (1:1 & 2:1).
Fig. 7: Correlation ratio by continuous variation (1:1 & 2:1).
Calculation of formation constant for the (FLU:BCP) complex:
The conditional stability constants (𝐾𝑓) of the ion-pair complexes were calculated from molecular ratio and the continuous variation
curves.
Data using the following equation12-13-14:
Where 𝐴 and 𝐴𝑚 are the observed
maximum absorbance and the absorbance value when all the Flucloxacillin sodium is
completely associated with Bromocresol purple, respectively. 𝐶𝑀 is the mole concentration of Flucloxacillin sodium at
the maximum absorbance and 𝑛 is the stoichiometry
with which dye ion associates with Flucloxacillin sodium. The log 𝐾𝑓 values for FLU-BCP ion-pair associates at correlation
ratio (1:1 & 2:1) by molecular ratio were 8.49 and 9.51 respectively, and by continuous
variation were 8.51 and 9.30 respectively.
Method’s
validation:
The
validity and suitability of the proposed method was assessed by linearity (evaluated
by regression equation), limit of detection (LOD), limit of quantification (LOQ),
accuracy (reported as percent %), precision (reported as RSD %), robustness, and
Sandellʹs sensitivity.
Linearity:
We studied the linearity of Flucloxacillin sodium standard
concentrations at the optimal conditions where we made a series of 10 mL of separated
volumetric flasks, each one contains 1 mL of BCP 5×10-3 M, and variable
concentrations of Flucloxacillin sodium stock solution 5×10-4 M, and
completed to 10 mL with Chloroform. Finally we measured the absorbance at 408 nm
for each concentration against the blank of BCP in Chloroform.
Fig. 8 presents the Flucloxacillin sodium spectra. The
range of linearity was obeyed to Beer’s law in concentration (2.47 – 22.23) μg/mL and the linearity
curve is presented in Fig. 9.
Limit
of Detection (LOD) and Limit of Quantification (LOQ):
In
spite of the measurement LOD and LOQ, five concentrations were analyzed in five
replicates.
LOD
and LOQ for Flucloxacillin sodium were calculated by using the following equations:
3.3 ×SD 10 ×SD
LOD
= –––––––; LOQ = ––––––––
m m
Where
SD, is the standard deviation of y intercepts of regression lines and m is the slope
of the calibration curve. The limit of detection (LOD) and limit of quantification
(LOQ) were to be 0.203 and 0.615 𝜇g/mL
respectively.
Fig. 8: spectra of (FLU -BCP):
C1:
2.47 𝜇g/mL, C2: 4.94 𝜇g/mL, C3: 7.41 𝜇g/mL, C4: 9.88 𝜇g/mL, C5:12.35 𝜇g/mL, C6: 14.82 𝜇g/ mL,
C7: 17.29 𝜇g/mL, C8: 19.76 𝜇g/mL, C9: 22.23 𝜇g/ mL.
Fig. 9: Calibration curve for (FLU-BCP),
n = 5 for each concentration.
Accuracy:
To determine the precision and accuracy of the proposed
method, five replicate determinations were carried out on three different concentrations
of standards (FLU). The precision and accuracy results are shown in table 1.
Table 1: Accuracy for determination of Flucloxacillin sodium.
|
material
|
Theoretical concentration (μg/mL)
|
 observed concentration (μg/mL)
|
SD µg/mL
|
Precision RSD
(%)
|
Accuracy (%)
|
|
Flucloxacillin sodium standard samples
|
2.47
|
2.40
|
0.03
|
1.25
|
97.17
|
|
7.41
|
7.33
|
0.11
|
1.50
|
98.92
|
|
12.35
|
12.48
|
0.21
|
1.68
|
101.05
|
|
17.29
|
17.34
|
0.17
|
0.98
|
100.29
|
|
22.23
|
22.18
|
0.08
|
0.36
|
99.78
|
: mean of five replicated determinations,
Accuracy (%) = (observed concentration/theoretical concentration) ˟
100,
Precision (RSD %) = (standard deviation/mean
concentration) ˟100.
Table
2: Intra-day precision for determination of Flucloxacillin sodium.
|
Found concentration μg/mL
|
Prepared concentration
𝜇g/mL
|
material
|
|
Precision
RSD %
|
*Time III
|
Precision
RSD %
|
*Time II
|
Precision
RSD %
|
*Time I
|
|
1.07
|
5.02
|
1.17
|
4.97
|
1.37
|
5.07
|
4.94
|
Flucloxacillin sodium
standard samples
|
|
1.78
|
9.81
|
1.30
|
10.02
|
1.81
|
9.94
|
9.88
|
|
0.76
|
15.00
|
1.40
|
14.75
|
1.98
|
14.69
|
14.82
|
*n =
5.
Table
3: Inter-day precision for determination of Flucloxacillin sodium.
|
Found Concentration μg/mL
|
Prepared concentration
𝜇g/mL
|
material
|
|
Precision
RSD %
|
*Day III
|
Precision
RSD %
|
*Day II
|
Precision
RSD %
|
*Day I
|
|
1.41
|
4.95
|
1.89
|
5.08
|
1.17
|
4.97
|
4.94
|
Flucloxacillin sodium
standard samples
|
|
1.40
|
9.85
|
1.51
|
9.81
|
1.30
|
10.02
|
9.88
|
|
2.73
|
14.83
|
1.00
|
14.95
|
1.40
|
14.75
|
14.82
|
*n =
5.
Table
4: Robustness test conditions used in this study.
|
Parameter
|
Measured deviation
|
*
µg/mL
|
RSD %
|
Percent (%)
|
*
µg/mL
|
RSD %
|
Percent (%)
|
*
µg/mL
|
RSD %
|
Percent (%)
|
|
Slit range
2 nm
|
2 nm
1 nm
|
4.98
4.93
|
1.08
2.11
|
100.81
99.80
|
7.33
7.51
|
1.24
1.78
|
98.92
101.35
|
9.99
10.04
|
0.66
0.37
|
101.11
101.62
|
|
scan speed medium
|
Fast
slow
|
4.98
4.92
|
1.08
1.05
|
100.81
99.60
|
7.33
7.55
|
1.24
2.35
|
98.92
101.89
|
9.99
9.90
|
0.66
0.95
|
101.11
100.20
|
|
Wavelength
|
+2 nm
-2 nm
|
4.87
4.87
|
0.92
1.56
|
98.60
98.60
|
7.29
7.31
|
1.87
2.09
|
98.38
98.65
|
9.89
9.83
|
1.67
1.60
|
100.10
99.49
|
*n = 5
Precision:
In
order to demonstrate the precision of the proposed methods, intra-day and inter-day
variability studies were performed at three different concentrations (4.94, 9.88,
and 14.82 𝜇g/mL) for Flucloxacillin sodium at the same day and also
at three different days. Method efficiency was tested in terms of RSD % for both
intra-day and inter-day precisions.
The
precision was ascertained by carrying out five replicates of standard Flucloxacillin
sodium under study and the mean was calculated. The results are showed in Tables
2 and 3. The RSD% results were not more than 2,73 during the determination in one
day or three days, where the method is considered very precise,
Robustness:
The
robustness of an analytical procedure is a measure of its capacity to maintain unaffected
results by a very small variation of some parameters and provides an indication
of its reliability during normal usage. The studied variables parameters were Slit,
scan speed and the wavelength which performed at three different concentrations
(4.94, 7.41, and 9.88 𝜇g/mL) for Flucloxacillin sodium. the results showed no
significant differences, Table 4.
Sensitivity
Sandell’s and molar absorptivity ε:
Sensitivity
of the proposed method for Flucloxacillin
sodium was determined by calculating Sandell’s
sensitivity (SS), it was to be SS = 0.0277 µg/cm2. The mean molar absorptivity
ε was found equal to 35671.7 L/mol.cm.
The
method parameters obtained during the study as: λ max, Linearity,
Stability of the complex, Solvent, Molar absorptivity ε, Regression equation, limit of
detection (LOD) and limit of quantification (LOQ), Sandell’s sensitivity
were calculated, where the absorbance measurements were obtained Beer’s law and
presented in table 5.
Table
5: Analytical parameters of Flucloxacillin sodium determination of standard solutions.
|
Parameters
|
Practical conditions
|
|
λ max (nm)
|
408
|
|
Linearity (μg/mL)
|
2.47 – 22.23
|
|
Stability of the complex
|
10 hours
|
|
Temperature of solution
|
25 ± 5 oC
|
|
Solvent
|
Chloroform
|
|
CBCP:CFLU, M
|
≥10
|
|
Molar absorptivity, ε (L/mol.cm)
|
35671.7
|
|
Regression equation
|
Y = 0.0701X + 0.0175
|
|
Slope (b)
|
0.0701
|
|
Intercept (C)
|
0.0175
|
|
R2
|
0.9999
|
|
LOD (µg/mL)
|
0.203
|
|
LOQ (µg/mL)
|
0.615
|
|
Sandell’ s sensitivity SS (µg/cm2)
|
0.0277
|
RAW
MATERIAL DETERMINATION:
We
determined Flucloxacillin sodium in raw material, imported from (RIBBON-China). The percentage was deduced from the precedent standard
curve. The obtained purity 99.00 %, was within the permissible limits set by the
USB legislation (USP34 NF 29 2011) not less than 95.00
% percent and not more than 102.00 %15.
CONCLUSION:
We
developed a new method which is suitable for the identification and quantification
of Flucloxacillin sodium in raw material. A good percentage shows that the method
can be successfully used in routine analyses. The proposed method is simple, sensitive,
rapid, specific, a little cost and could be applied for quality control of Flucloxacillin
sodium. The levels of Flucloxacillin sodium in raw material were conform to USB permissible limits. The method
can be also proposed to determine Flucloxacillin sodium in pharmaceutical formulation.
ACKNOWLEDGEMENT:
The
Ministry of High Education in Syria financially and technically supported this work
through department of Chemistry, Faculty of Science, University of Aleppo, Syria.
REFERENCE:
1.
Attia K, Abdel-Aziz
O, Magdy N, Mohamed G. Determination of Flucloxacillin-sodium in Binary Mixture
with Ampicillin-trihydrate Using Univariate and Multivariate Spectrophotometric
Methods: A Comparative Study. Journal of Advanced Ph. 2018; 2 (3): 180-190.
2.
Eissa M SH,
Darweish E, Elghobashy M, Shehata M. Rapid Selective TLC-Densitometry Method for
Simultaneous Determination of Amoxicillin and Flucloxacillin in Their Pure Forms
or in Their Pharmaceutical Preparation. Analytical Chemistry Letters. 2018; 8 (2): 188-194.
3.
Konaria S,
Jacob J. Stability-indicating LC-Analytical
Method Development and Validation for The Simultaneous Estimation of Flucloxacillin
and Amoxicillin in Pharmaceutical Dosage Form. Journal of Taibah Univ for Science. 2015;
9: 167–176.
4.
Vijayalakshmi R, Sowjanya
D, Archana S, Dhanaraju MD. Application of Extractive Spectrophotometric Methods
for the Determination of Flucloxacillin and Trandolapril Using Bromocresol Green
Asian Journal of Pharmaceutical and Clinical Research. 2014; 7(4): 216-218.
5. Lakshmi R. V, Kiranmai K., Dhanaraju M D. Spectrophotometric
Determination of Flucloxacillin Sodium Using Folin-Ciocalteu Reagent. Oriental Journal
of Chemistry. 2010; 26(3): 35-1138.
6. Gujral R, Haque S, Shanker P. A Sensitive Validated Spectrophotometric
Method for the Determination of Flucloxacillin Sodium. E-Journal of Chemistry. 2009;
6(S1): S397-S405.
7. Prakash V, Konari Sh N, Suresh G. Development and Validation
of Spectrophotometric Method for The Estimation of Flucloxacillin. IJPSR. (2012);
3(6): 1806-1808.
8. Attia K A M, Nassar M W I, El-Zeiny M B b, Serag A. Different
Spectrophotometric Methods Applied for The Analysis of Binary Mixture of Flucloxacillin
and Amoxicillin. A comparative study Spectrochimica Acta Part A Molecular and Biomolecular
Spectroscopy. (2016); V 161: 64–69.
9. Nikam D S, Bonde Ch G, Surana S J, Venkateshwarlu G, Dekate
P G. Development and Validation of RP-HPLC Method for Simultaneous Estimation of
Amoxicillin Trihydrate and Flucloxacillin Sodium in Capsule Dosage Form. International
Journal of Pharm Tech Research. 2009;1(3): 935-939.
10. Sankar D G, Rajeswari A, Babu A N, Krishna M. RP-HPLC Method
for Estimation of Flucloxacillin Magnesium and Sodium Benzoate in Oral Suspension.
Asian Journal of Chemistry. 2009; 21(8): 5843-5846.
11. Michael W K, Bright S A, David N M. A Simple Validated RP-
HPLC Method for The Analysis of Flucloxacillin Sodium in Capsule Dosage Form. World
Journal of Pharmacy and Pharmaceutical Sciences. 2016; 5(4): 499-508.
12. Kudige N P, Kanakapura B, Madihalli S R. Simple and Selective
Spectrophotometric Determination of Ofloxacin in Pharmaceutical Formulations Using
Two Sulphonphthalein Acid Dyes. Hindawi Publishing Corporation.2013.
13.
Amina A S, Goudab A
El-F, El-Sheikh R b, Zahran F. Spectrophotometric Determination of Gatifloxacin
in Pure Form and in Pharmaceutical Formulation. Spectrochimica Acta Part A. 2007;
(67): 1306–1312.
14.
Britton H.T.S., Hydrogen
Ions, fourth ed., Chapman and Hall, 1952.
15.
Pharmacopeia USP34 NF 29 2011.