1. INTRODUCTION:
Development of analytical methods that save time and solvents
and provide accurate and precise analysis for commercial products in the field of
industrial laboratories is an interesting subject for analytical chemists, who focus
on using UV-Visible spectrophotometric method which is considered one of the most
popular and cheapest techniques1.
Unfortunately, classical spectrophotometric methods have
many disadvantages i.e. low selectivity, caused by overlapping spectra. Analytical
chemists have always been trying to solve these problems by developing new analytical
methods or approaches to increase selectivity. One of these is derivative spectrophotometry
(DS)1. DS is one of the advanced modern spectrophotometric techniques,
utilized for extracting qualitative and quantitative information from spectra of
unresolved bands2. The derivatization of zero-order spectrum may lead
to separation of overlapped signals, elimination of background caused by presence
of other compounds in a sample3. These properties can allow the quantification
of one or few analytes without initial separation or purification3. DS
may be an alternative to hyphenated analytical instrumentations or techniques such
as HPLC, LC–MS, GC–MS, and LC–NMR which always require a prior step such as extraction
and other tedious analytical process during analysis, Hence, this technique is economic,
simple to apply, very rapid and affordable for analysis of any mixture4.
Taking into account all the above advantages, DS has been applied in the analysis
of different pharmaceuticals, food, biological samples, cosmetics and environmental
samples which contain many analytes without any interferences5-12, for
both stability studies and multicomponent analysis13-18. This technique
can be used for all pharmaceutical drugs, especially antibiotic mixtures which have
pharmaceutical importance and a common use to treat bacterial infections19.
Hence, this work focused on analyzing one of the common-used antibiotics worldwide,
Augmentin, which consists of amoxicillin (beta-lactam antibiotic), Figure 1a, and
clavulanic acid (beta-lactamase inhibitor), Figure 1b19. This binary
mixture has overlapping spectra in zero order (absorbance). Therefore, the aim of
this study was to develop derivative UV spectrophotometric method for simultaneous
determination of amoxicillin and clavulanic acid in their tablets (Augmentin 1000mg),
other previous works focused on 625mg tablets, using water as solvent. This work
used derivatization's parameters (d𝛌 and scaling factor) to enhance results of derivatization
to avoid problems related to differences of drug’s concentrations in tablets. In
addition, this work compared proposed method with reported chromatographic methods
(HPLC) and confirmed that the developed method has the same accuracy and precision
of reference method.
Figure 1: (a) Amoxicillin and (b) Clavulanate potassium
2. MATERIALS AND METHODS:
2.1 Materials
Pure materials: Amoxicillin trihydrate (Amox) and Clavulanate
Potassium (Clav K) containing the microcrystalline cellulose (MCC) 1:1 weight ratio
were kindly supplied by Maatouk Pharma.
Samples (Pharmaceutical formulation): Augmentin coated
tablets (875mg Amox and 125mg Clav), manufactured by GlaxoSmithKline for Pharmaceutical
Industries, (United Kingdom).
Methanol HPLC grade (Merck, Germany), Monobasic Sodium
phosphate (Avonchem, UK) and distilled water.
2.2 Methods:
2.2.1 Instruments:
Spectral procedure: The absorption measurement was carried
out on a double beam UV-Visible spectrophotometer (Shimadzu, Japan), model UV-1800
with 1.00cm quartz cells. Handling of data was achieved by using the UV-PC software.
Chromatographic separation: High performance liquid chromatography
(HPLC) analysis was performed on a Shimadzu (japan). The procedure was done according
to USP 2018 (column C18: 30cm × 4mm; 5µm, detector: UV 200nm, flow rate: 2ml/min,
injection size 20µl, mobile phase: mixture of methanol and buffer (phosphate buffer
with pH = 4.4±0.1) in a ratio of (1:19 v/v) respectively, isocratic mode).
2.2.2 Prepared solutions:
2.2.2.1 Preparation of Standard Solutions:
Stock solutions (0.4mg/ml) were prepared by dissolving
40mg of Amox and 80mg of Clav/MCC into two separate 100ml volumetric flasks consisting
of 70ml of distilled water using ultrasonic devise. The final volume was adjusted
to 100ml. A standard series of the solutions containing (5-45μg/ml) of Amox
and (1-10μg/ml) of Clav were prepared from the stock solutions, and all dilutions
were freshly made to avoid instability problem.
2.2.2.2 Preparation of Laboratory mixtures:
Laboratory mixtures containing different ratios of the
two drugs were prepared by transferring different aliquots of Amox and Clav from
their stock solutions (0.4mg/ml) separately into a set of 10ml volumetric flasks
then the volume was adjusted with distilled water.
2.2.2.3 Preparation of Commercial drugs solutions:
Ten tablets were weighted and powdered then an average
weight of one tablet was weighted and dissolved in 1000ml volumetric flask (consisting
of 900ml of distilled water with 30 mins of mechanically shaking) and adjusted to
1000ml. The solution was then filtered through 0.45μm disposable membrane filter
then the final solution was diluted to ratio (2:50 v/v)..
3. PROCEDURE:
3.1 Method Development
Zero-order (absorptionDo) spectra of the two
drugs were recorded at (200 – 400nm) against distilled water as a blank. First order
(derivativeD1) spectra were obtained for all Do spectra using
the software (UV Probe - [Spectrum]). The optimization parameters (Δ𝛌 and scaling factor) were set by testing different
values of Δ𝛌
(1, 2, 4) and scaling factors (1, 5, 10, 15) by using standard solutions. Measurements
were calculated at zero crossing points (peak amplitude of Amox spectra at zero
crossings for Clav and peak amplitude of Clav spectra at zero crossings for Amox).
The calibration curves between the amplitude values against the corresponding concentration
of Amox and Clav were separately constructed and respective regression equations
were computed. Determination of zero-crossing points was obtained using standard
solutions. However, Laboratory mixtures were prepared for studying the accuracy
of zero-crossing points and determining the two drugs simultaneously without any
interference.
3.2
Method Validation:
Method validation was performed following ICH specifications
for linearity, detection limit, quantitation limit, precision and accuracy. Standard
solutions were used for studying (linearity, detection limit, quantitation
limit). Precision and accuracy (possibility of simultaneous determination without
separation) were undertaken by using Laboratory mixtures. However, commercial solutions
were implemented for testing the accuracy of the proposed method (apart from the
excipients that do not influence upon the analysis).
3.3 Statistical analysis:
The applicability of the validated method was tested by
analyzing the pharmaceutical dosage form. The same solution was analyzed by the
developed method and via HPLC technique. The results obtained from derivative method
(n=6) were compared with those obtained from HPLC method (n=6). The comparison was
then made regarding the precision (Fisher test) and accuracy (t-test).
4. RESULTS AND DISCUSSION:
4.1 Method development:
The UV absorption spectra for Amox and Clav are shown in
Figure 2. It shows that absorption spectra of Clav were largely overlapped by absorption
spectra of Amox. Therefore, it may be difficult to use direct UV spectrophotometry
for simultaneous determination of Clav and Amox without prior separation.
Figure 2: Zero order spectra of Amox 20 μg/ml,
Clav 5 μg/ml (b), and mixture (20+5) μg/ml (c).
Hence, derivative UV spectrophotometric method was developed
to determine Amox and Clav in their formulations. The derivatization of UV spectra
removed the overlapping, making it possible to determine the analytes in the presence
of excipients (Figure 3).
Figure 3: First order (Derivative) spectra of Amox
(5-45 μg/ml), and Clav (1-10 μg/ml).
Many derivatizations’ parameters were optimized. Firstly,
Δλ which plays a major role in enhancing the signal-to-noise
ratio, so that the noise can be significantly lowered without loss of the signal
of interest20. Figure (4) shows that Δλ =2 and 4 were suitable for Amox. However, as
observed in figure (5) only Δλ= 4 worked for Clav. As a result, the best value of this
parameter was (4) for both components.
Figure 4: Influence of Δλ values on the shape of Amox's D1 spectra,
enlarged part shows the noise by changing Δλ.
Figure 5: Influence of Δλ values on the shape of Clav's D1 spectra.
Secondly, scaling factor was the other parameter. This factor proportionally affects the value of signal’s derivative (amplitude). hence, it is useful for enhancing weak signals, which may be produced by a low‑concentration component21. As mentioned, this work concerned the analysis of Augmentin tablets, which composed of Clavulanate (Clav) as a minor and co-formulated component with Amoxicillin (Amox) in ratio (1:7), this formulation shows the importance of using scaling factor in this research. This parameter also played a role in solving the dilution’sproblem of commercial Augmentin samples; when dilution ratio is above (10:100 v/v of stock solution 1000 ml), it gives a spectrum distortion at the same region that will be used for measurement (Figure (6)). All above has been affected linearity range of Clav. The range (1 - 10 μg/ml) was suitable to determinate Clav in tablets (1000 mg). Hence, it was necessary to optimize this parameter in this work. The value = 15 was selected in this case for both drugs to increase the amplitude of first derivative spectra for Clav mainly, with respect to Amox’s linearity, (Figure (7)).
Figure 6: The shape spectrum of
first derivative for commercial samples in
cases of
dilution ratio above 10:100 v/v
Figure 7: Influence of
scaling factor on amplitude’s value of
Amox 45
µg/ml (A), and Clav 10 µg/ml (B).
"Zero crossing"technique22 was applied for the determination
of these drugsin their formulations, Figure (8). Amox could be analyzed by measuring
its first derivative amplitude at zero crossing point of Clav at 278.5nm. In addition,
Clav could be determined at zero crossing points of Amox at (219.5 and 228.3) nm.
Figure 8: The first-order derivative spectra of Amox, Clav, and a mixture of Amox and Clav
However,
228.3 nm was chosen for the determination of Clav because it showed significant recovery of Clav in the laboratory mixtures with low relative error when compared to amplitude measured at 219.5 nm, as shown in table 1.
Table 1: Recovery and relative error for the determination
of Clav at 219.5nm and 283.5 nm.
|
Wavelength
(nm)
|
Recovery
|
Relative Error
|
|
|
SD
|
|
SD
|
|
219.5
|
69.951
|
20.36
|
-30.049
|
20.36
|
|
228.3
|
98.53
|
0.81
|
-1.47
|
0.81
|
The peak amplitude was measured at 278.5nm at different concentrations of Amox, and a calibration curve between amplitude versus concentration of Amox was constructed. Alternatively, the linearity equation was obtained from the calibration curve. The same procedure was done for Clav at 228.3nm.
4.2 Method Validation:
Method validation was performed according to ICH guidelines
(23) for the developed method as follows:
4.2.1 Linearity:
Linearity range was determined for both analytes for analytical
method by analyzing the different concentration solutions. Amox exhibited excellent
linearity in the range of 5 to 45 μg/ml with excellent correlation coefficient
(r2=0.9998). Clav was linear in the range of 1 to 10 μg/ml with
a good correlation coefficient (r2=0.9986). The linearity range, regression
equations, and correlation coefficients are tabulated in Table 2.
4.2.2 Limit of Detection (LOD) and Limit of
Quantification (LOQ):
LOD and LOQ were determined according to the ICH recommendations,
Table 2. The approach based on the standard deviation (SD) of the intercepts and
the slope was used for determining the LOD and LOQ, as shown in equations 1 and
2.
|
LOD=3.3×SD / slope
|
(1)
|
|
LOQ=10×SD / slope
|
(2)
|
Table 2: Regression equations and validation parameters
results for Amox and Clav
|
Parameter
|
Amox
|
Clav
|
|
Measurement
wavelength
|
278.5 nm
|
228.3 nm
|
|
Range μg/ml
|
5-45
|
1-10
|
|
LOD μg/ml
|
0.1768
|
0.1693
|
|
LOQ μg/ml
|
0.5357
|
0.5131
|
|
Regression
equation
|
Y = -0.0013 x -
0.0004
|
Y = -0.0178 x -
0.0008
|
|
determination
coefficient (r2)
|
0.9998
|
0.9986
|
|
*Intraday(RSD%)
|
1.506
|
1.73
|
|
**Interday
(RSD%)
|
0.379
|
1.59
|
*The intraday precision, Average of three experiments
within day.
**The interday precision, Average of three experiments
in three successive days.
4.2.3 Precision:
The intraday and interday precision of the proposed method
were determined. For intra-day precision, three different concentrations of both
analytes, within the linearity range, were analyzed by the optimized method in triplicate
on the same day. For interday precision, fresh preparations with the same concentrations
were analyzed for three successive days. Relative standard deviation (RSD%) was
calculated as shown in Table 2. The low % RSD (<2.0) indicate good precision.
4.2.4 Accuracy:
Accuracy was determined by comparing measured concentrations
of Amox and Clav with the actual values. The accuracy of the results was checked
by applying the proposed method for the determination of laboratory prepared mixtures
containing both drugs within the linearity range. The concentrations were obtained
from the corresponding regression equations, and the percent recoveries were calculated
as shown in Table 3. Good recoveries indicated good accuracy and there was no interference
between the two drugs.
Table 3: Determination of Amox and Clav in Laboratory
Prepared Mixtures by the Proposed Method
|
Mixture number
|
Claimed taken (μg/ml)
|
DD1
Method Recovery %
|
|
|
Amox
|
Clav
|
Amox
|
Clav
|
|
1
|
20
|
5
|
99.88
|
102.47
|
|
2
|
30
|
6
|
98.71
|
99.44
|
|
3
|
40
|
7
|
99.63
|
101.2
|
The accuracy of the developed method was further investigated
using the standard addition technique, which was performed by adding known amounts
of pure Amox (0.2, 0.3, 0.4) mg/ml and Clav (0.08, 0.12, 0.16) mg/ml separately
to the previously analyzed pharmaceutical preparation. The total amount of Amox
and Clav was then determined using the corresponding regression equations, and the
percent recoveries were calculated, Table 4. This showed good accuracy of the developed
method and that the excipients of the tablets did not interfere with the analysis
of the two compounds, as a result the excipients didn’t have a negative effect to
the analysis.
Table 4: Determination of Amox and Clav in Augmentin
tablets by DD1 Spectrophotometric Method (the Standard Addition
Technique).
|
Product
|
|
|
DD1 Method
|
|
Augmentin
|
Taken (mg/ml)
|
Added (mg/ml)
|
Found (mg/ml)
|
Recovery %
|
|
Amox
|
0.0963
|
0.2
|
0.278
|
93.83
|
|
0.3
|
0.415
|
104.95
|
|
0.4
|
0.494
|
99.54
|
|
|
|
|
 ± SD
|
99.44 ± 5.56
|
|
Clav
|
0.021
|
0.08
|
0.1006
|
99.49
|
|
0.12
|
0.1407
|
99.65
|
|
0.16
|
0.1787
|
98.67
|
|
|
|
|
 ± SD
|
±0.43
|
4.3 Statistical analysis:
Table 5 shows statistical comparisons of the results obtained
by both the proposed and reported methods. The calculated t and F values were less
than the tabulated ones, so there was no significant difference between the proposed
and the reported methods with respect to accuracy and precision.
Table 5: Statistical comparison of the results obtained
by the proposed spectrophotometric method and the reported method for the
determination of Amox and Clav in dosage form.
|
Items
|
D1Method
|
Reported Method
(HPLC)
|
|
Amox
|
Clav
|
Amox
|
Clav
|
|
*Mean Recovery
|
98.32
|
136.56
|
97.68
|
135.02
|
|
SD
|
1.187
|
3.95
|
0.567
|
2.09
|
|
N
|
6
|
6
|
6
|
6
|
|
**p-value
|
0.273
|
0.358
|
-
|
-
|
|
**Student's t
test
|
1.232
|
1.011
|
-
|
-
|
|
**F test
|
4.38
|
3.552
|
-
|
-
|
* Mean Recoveries of six determinations for the
proposed method. Recoveries were calculated considering the labeled amount reported
by the manufacturer.
** the tabulated tvalue at 95% confidence limit for 5
degrees of freedom (n =6) is 2.571, the tabulated Fvalue at 95% confidence
limit for 5 degrees of freedom for the proposed methods is 5.05, and the p‑value
= 5%
5. CONCLUSION:
UV spectroscopic method (1st derivative) was
developed for simultaneous determination of Amox and Clav in tablets. This method
is simple, rapid, accurate, precise, economic and eco-friendly by using water as
solvent. The analytical method involved optimization of derivatization's parameters
to obtain best signals with lowest noise. First derivative spectroscopic method
can determine both the analytes in a binary mixture in two steps: derivatization
and measurement of amplitude at zero-crossing wavelength. The statistical comparison
results confirmed that there were no significant differences between the reported
method (HPLC) and the proposed technique. To conclude, the proposed method was successfully
applied for the routine analysis of binary mixtures, both in bulk powders and in
dosage forms, in quality control laboratories without any preliminary separation
steps.
6. ACKNOWLEDGMENTS:
The authors would like to thank Maatouk Pharma for pharmaceutical
industries (Damascus, Syria) for providing pure materials and Golden Med Pharma
for pharmaceutical industries (Tartous, Syria) for providing laboratory facilities
in order to carry out this study.
7. REFERENCES:
1.
Karpinska J. Basic
principles and analytical application of derivative spectrophotometry. Macro to
nano spectroscopy, book edited by Jamal Uddin. 2012 Jun 29:253-68. doi:
10.5772/37673.
2.
Rojas FS, Ojeda CB,
Pavon JC. Derivative ultraviolet—visible region absorption spectrophotometry
and its analytical applications. Talanta. 1988 Jun 21;35(10):753-61. doi:
10.1016/0039-9140(88)80179-6.
3.
Patel KN, Patel JK,
Rajput GC, Rajgor NB. Derivative spectrometry method for chemical analysis: A
review. Der Pharmacia Lettre. 2010;2(2):139-50.
4.
Lotfy HM, Saleh SS.
Recent development in ultraviolet spectrophotometry through the last decade
(2006-2016): A review. International Journal of Pharmacy and Pharmaceutical
Sciences. 2016 Oct 1;8(10). doi: 10.22159/IJPPS.2016V8I10.13537.
5.
Jones K, Sweeney GD.
Quantitation of urinary porphyrins by use of second-derivative spectroscopy.
Clinical Chemistry. 1979 Jan 1;25(1):71-4. doi: 10.1093/clinchem/25.1.71.
6.
Altιnöz S, Toptan
S. Determination of Tartrazine and Ponceau-4R in various food samples by
Vierordt's method and ratio spectra first-order derivative UV
spectrophotometry. Journal of food composition analysis. 2002;15(6):667-83.
doi: 10.1006/jfca.2002.1072.
7.
Ch R, T B, Babu R.
Development and Validation of Zero and First Order Derivative
UV-Spectrophotometric Method for Determination of Febuxostat in Bulk and in
Formulation. Research Journal of Pharmacy and Technology. 2013 Feb
1;6(2):208-11. doi: 10.13140/RG.2.2.17671.42404.
8.
Dekhil AB, Ghorbel A,
Boubacker T. Direct determination of nitrate in natural water by ultraviolet
first derivative spectrophotometry. Analytical CHEMISTRY An Indian Journal.
2014 Feb 1.
9.
Teja BR, Varma BRK,
Chandaka Prasanna Kumar, Annapurna MM. First order derivative
spectrophotometric methods for the determination of Fluorometholone in
ophthalmic preparations. Research Journal of Pharmacy and Technology. 2017 Jul
17;10(4):1145-8. doi: 10.5958/0974-360X.2017.00206.2.
10.
Rele RV. UV Derivative
Spectrophotometric Methods for validation of Esomeprazole Magnesium tri-hydrate
in Bulk and Pharmaceutical Dosage Form. Research Journal of Pharmacy and
Technology. 2018 Jun 12;11(1):135-8. doi: 10.5958/0974-360X.2018.00026.4.
11.
Annapurna MM, Sheela AS,
Yasaswini RS. New first derivative spectrophotometric methods for the
estimation of Bumetanide in tablet dosage forms. Research Journal of Pharmacy
and Technology. 2019 Dec 24;12(10):4790-4. doi: 10.5958/0974-360X.2019.00827.8.
12.
Sushma G, D SH,
Mukthinuthalapati MA. New first derivative spectrophotometric methods for the
determination of Sitagliptin - An antidiabetic agent. Research Journal of
Pharmacy and Technology. 2020 Jul 4;13(6):2838-42. doi:
10.5958/0974-360X.2020.00505.3.
13.
Patil VD, Bhadoriya AS,
Ingale KD, Chabukswar AR, Choudhari VP, Kuchekar BS. Development and Validation
of Ratio Spectra Derivative Spectrophotometric Method for Determination of
Mefenamic acid and Ethamsylate in combined Formulation. Research Journal of
Pharmacy and Technology. 2010;3(3):921-3.
14.
Chabukswar AR, Tambe SD,
Choudhari VP, Shailesh N. Sharma, Mohokar MN, Chate S. Ratio Derivative
Spectrophotometry Method for Simultaneous Estimation of Metoprolol and
Amlodipine in their Combined Dosage Form. Research Journal of Pharmacy and Technology.
2012 Jan 1;5(7):950-4.
15.
Veeranna V, Rao VS,
Laxmi VV, R. VT. Simultaneous Second Order Derivative Spectrophotometric
Determination of Cadmium and Cobalt using Furfuraldehyde Thiosemicarbazone
(FFTSC). Research Journal of Pharmacy and Technology. 2013 Jan 1;6(5):577-84.
16.
Abdelraoof A.
Spectrophotometric methods for the determination of trazodone hydrochloride in
presence of its alkaline degradation product. Innoriginal: International
Journal of Sciences. 2017 Sep-Oct:5-11.
17.
Khayata W, Zakri DA. Two
simple spectrophotometric methods for the simultaneous determination of
amoxicillin trihydrates and flucloxacillin sodium. Research Journal of Pharmacy
and Technology. 2017 May 1;10(5):1327-32. doi: 10.5958/0974-360X.2017.00235.9.
18.
Rele RV. Derivative
Spectrophotometric Estimation of Amoxicillin Trihydrate and Carbocisteine by
Third Order Derivative Spectroscopy method in Combined Dosage Form. Research
Journal of Pharmacy and Technology. 2017 Sep 22;10(6):1758-61. doi:
10.5958/0974-360X.2017.00310.9.
19.
Atici EB, Yazar Y,
Ağtaş Ç, Ridvanoğlu N, Karlığa B. Development and
validation of stability indicating HPLC methods for related substances and
assay analyses of amoxicillin and potassium clavulanate mixtures. Journal of
Pharmaceutical Biomedical Analysis. 2017 Mar 20;136:1-9. doi:
10.1016/j.jpba.2016.12.032.
20.
Ozdemir A, Korkmaz A.
Comparative study of electrospray mass spectrometry and first derivative method
and validation by HPLC method. Journal of Food and Drug Analysis.
2007;15(2):118. doi: 10.38212/2224-6614.2434.
21.
Attimarad M, Venugopala
KN, Aldhubiab BE, Nair AB, SreeHarsha N, Pottathil S, et al. Development of UV
spectrophotometric procedures for determination of amlodipine and celecoxib in
formulation: use of scaling factor to improve the sensitivity. Journal of
Spectroscopy. 2019 Dec 7;2019. doi: 10.1155/2019/8202160.
22.
Kus S, Marczenko Z,
Obarski N. Derivative UV-VIS spectrophotometry in analytical chemistry. Chem
Anal. 1996;41(6):889-927.
23.
International Conference
on Harmonization (ICH), Q2B: Validation of Analytical Procedures: Methodology,
62. US FDA, Federal Register; 1997.
=0.9998) and (1 - 10μg/ml with