INTRODUCTION:
Ketoconazole
[(±)-cis-1-acetyl-4-(4-{[2-(2,4-
dichlorophenyl)-2-(1H-imidazol-1-ylmethyl)-1,3-dioxolan- 4-yl] methoxy} phenyl)
piperazine] (Figure 1) is imidazole derivate that have indicate as antifungal
treatment 1. Ketoconazole has two pH values (both alkaline),
which are 2.94 from imidazole groups and 6.51 from piperazine groups 2. Ketoconazole is a biopharmaceutics classification
system (BCS) class II, meaning low solubility and high permeability 3. The solubility of ketoconazole is very slightly
soluble (0.017 mg/mL at 250C), insoluble at neutral pH, and slightly
soluble in acidic solution. The low solubility and rate of dissolution lead to
reduce the release of ketoconazole so that the effectivity of ketoconazole
decrease 4.
Figure 1. Ketoconazole Structure
Ketoconazole is a weakly basic drug that
has lower solubility at higher pH. The aqueous environment of the
gastrointestinal tract in the small intestine has gradually pH value range of
5.5-8.3, which is the solubility of ketoconazole will decrease in this
condition5. Determination of solubility of ketoconazole in the
base solution is not easy due to the poor solubility. Several studies have been
reported about the solubility of ketoconazole in simulated intestinal medium 6–9 that the medium need complex compositions. A simple
medium such as buffer pH 6.8 that the pH same with a pH value of simulated
intestinal medium can be used to represent solubility ketoconazole in
intestinal fluid. The quantification of ketoconazole can be determined by
several analytical methods, such as HPLC, Spectrophmetry, Colorimetry,
Voltammetric, Electroxidation 10. The measurement of ketoconazole using a
spectrophotometer has been used by Shrivastava, et al (2020), Kansagra, et al
(2013), Popovska, et al (2014), and Saez, et al (2018). The Spectrophotometry
UV have some advantage to analysis concentration of the drug, includes simple,
rapid, low cost, efficient, less time-consuming sample preparation, minimal
maintenance problems than other instrumental techniques 11,12. Several studies have been developed
spectrophotometric methods to determine concentration of drugs, such as to
analysis esomeprazole in tablet 13, nimesulide and diclofenac sodium in dosage form 14, darifenacin hydrobromid in bulk and tablet dosage
form15, choline bitartrate in bulk and tablet 16, atenolol and ivabradine HCl 17, eprosartan mesylate in bulk and dosage form 18, benfotiamine in bulk and dosage form 19, ursodeoxycholic acid in bulk 20, lurasidone in bulk and dosage form 21, cefuroxime axecil in bulk and dosage form 22, etc.
The objective of this research is to
validate the measurement method of solubility ketoconazole in base solution
using a spectrophotometer.
MATERIALS AND METHODS:
Materials:
Ketoconazole from PT. Kimia Farma,
Indonesia, deionized water was supplied from CV. Alfa Kimia, methanol,
potassium dihydrogen phosphate (KH2PO4), sodium hydroxide
(NaOH) were purchase from Sigma-Aldrich.
Preparation Phosphate Buffer Solution pH
6.8:
Phosphate Buffer was prepared by mixed 250.0 mL KH2PO4
solution (6.8 gram in 250.0 mL deionized water) and 125.0 mL NaOH (2.0
gram in 250.0 mL deionized water), then diluted to 1000 mL deionized water.
NaOH has been used to reach pH 6.8.
Solubility studies:
The equilibrium solubility of ketoconazole was
determined by the shake flask method. An excess amount of ketoconazole (1.0 g)
was added to 10.0 mL buffer pH 6.8, then shaker in shaking water bath at 370C
and 100 rpm. Aliquots were withdrawn at the end of the experiment (24 h) as
equilibrium reached then centrifuged at 10.000 x g for 30 min. Taking the
supernatant and then determined the concentration of ketoconazole by
spectrophotometer UV. Assays were performed in triplicate.
Preparation of stock solution:
A stock solution of ketoconazole was prepared by
dissolving 20.0 mg ketoconazole in 100.0 mL methanol to obtain the final
concentration which was 200 ppm. The spectrum of absorption maximum (λmax)
was determined by measure the sample that was run from 200-400 nm. Then the
solution was sonicated to dissolve and remove air completely.
Linearity:
Linearity was performed by 6 series of standard
solution with concentration 5, 10, 20, 40, and 50 ppm as follows: from standard
stock solution, appropriate aliquots 0.25, 0.5, 1.0, 2.0, and 2.5 mL were
pipette out in 10.0 mL volumetric flasks and diluted by buffer pH 6.8. The
calibration curve was plotted using concentration and absorbance. Linearity was
evaluated by linear regression analysis.
Precision:
Precision was determined by repeatability (intra-day)
and intermediated precision (inter-day). In intraday, precision was assessed by
analyzing 3 standard solutions at concentrations 10, 20, and 40 ppm that were
measure three times a day.
Accuracy:
Accuracy was determined by the standard addition
method at three levels (80%, 100%, and 120%). The standard addition method was
prepared by adding an appropriate amount of stock ketoconazole standard (1.6,
2.0, and 2.4 mL) to samples (1.0 mL) and made up to volume ad 5.0 mL using
buffer pH 6.8. The recovery was carried out by calculating the ratio of the
total amount of sample and the additional standard solution comparing with the
concentration sample without a standard solution.
Limit of detection (LoD) and quantification (LoQ):
LoD and LoQ were found by successive dilution of
standard solution (5, 10, 20, 40, and 50 ppm). The LoD represents the lowest
amount of analyte in the sample which can be detected that was defined as the
analyte concentration that gives a signal equal to yb + 3.3sb,
where yb is the signal of the blank and Sb
is the standard deviation. The LoQ represents the lowest amount of analyte
which can be quantitatively determined that was defined yb +
10Sb.
RESULT AND DISCUSSION:
Selection of
Suitable Wavelength:
A linearity
correlation was determined at λmax which was the λmax
was found at 207, 230, and 289 nm using buffer pH 7.8 as a blank. The λmax
which be chosen was 230 nm rather than either 207 nm or 289 nm. The wavelength
at 207 nm was suspected as a solvent absorption region. If the λmax used
that the solvent absorbs the radiation too, the result may be not accurate due
to is not only sample measured but also solvent.
The wavelength at 289 nm has molar
absorptivity (Ꜫ) lower than at 230 nm. The molar absorptivity is defined
by Beer’s law as Equation 1.
(1)
where A is absorbance, Ꜫ is molar
absorptivity (M-1cm-1), b is the path length of the
cuvette (usually 1 cm), and c is the concentration of the solution (M) 23.
However, the absorbance error can occur in
absorbance maximum due to a systematic bias in wavelength setting (function of
the width of the absorption band of target substance). The systematic error
sources are from solution temperature, the volume of solute apparent, buoyancy
correction, cell orientation, multiple reflections, finite slit width, stray
radiation, and wavelength offset 24
Linearity:
The
recommendation minimal concentration of standard solution in ICH Q2, WHO, and
AOAC recommended minimum 5 concentration 25,26, whereas IUPAC recommended minimum 627. In this study, the concentration used 5 values of
concentration, there were 5, 10, 20, 40, and 50 ppm. Range concentration of
calibration standard in IUPAC27 is 0-150% or 50-150% of concentration from target
level, whereas in ICH Q2 for assay of active substance is normally 80-120%, for
content uniformity is 70-130%, for dissolution test is ±20% over the specified
range26. The target result of assay ketoconazole in this
study was around 20 ppm so that the range of standard curve that have been
chosen was 5-50 ppm have been appropriate 28.
The calibration curve was shown in Figure 2
using standard calibration. The linearity result was described by the
regression equation: y=a+bx, where y is absorbance, x is the concentration
(ppm). Evaluation of linearity was determined by intercept (a), slope (b), and
coefficient (r2).
Figure 2 showed the regression equation to
be y=0.0448x-0.13. The value of the correlation coefficient has been found
0.9994 which represents the method was verified linear within a concentration
in the range of 5-50 ppm that met the Lambert-Beer Law 29,30. The slope value can show a sensitivity of method
that expressed as molar absorptivity (Ꜫ) 29. The yield of molar absorptivity ketoconazole that
has been calculated from slope value was 23801 M-1cm-1.
Figure 2. Calibration curve of ketoconazole at 230
nm
Accuracy:
The accuracy is
sometimes defined as trueness that expresses the closeness of agreement between
the value which is accepted either as a conventional true value or an accepted
reference value and the value found 26.
The accuracy of the method was evaluated in
the standard addition method at 3 levels with 3 replicates. Result within the
range 93.84-95.95% that presented in Table 2. The %recovery was acceptable that
was 80-110% for concentration analyte with 10 ppm according to the AOAC
standard of the acceptable mean recovery 31. This result not met similar result with other
studies which have been developed previously that using tablet as matrix
(96-104%)28. The higher the amount of analyte in matrix, the
higher accuracy can be obtained. Ketoconazole in tablet could be detected
concentration more than 90%, whereas concentration ketoconazole in buffer
alkali solution was very low.
Precision:
The precision is
the degree of agreement among individual result from multiple sampling of the
homogeneous sample. It should be measured by the scatter of individual result
from the mean and expressed as the relative standard deviation (RSD) 25. Precision may be considered at three levels, which
are repeatability, intermediate precision, and reproducibility 26. In this study, the level precision that have been
used was intermediate precision with repeatability for each sample. The
intermediate precision express with different days, which were intraday (same
day) and inter day (3 different days consecutively).
The intraday and interday precision study
results are summarized in Table 3 and Table 4. The relative standard deviation
(%RSD) value intraday was found 0.267, 0.112, and 1.237. The relative standard
deviation (%RSD) value interday was found 0.296, 0.008, and 0.792. These values
were less than 2% for intraday and interday, indicating the method is good
precision. When a RSD precision is expressed, the corresponding variance is
used (e.g repeatability or intermediate precision). The computed RSD is
therefore the ratio of two random variables, giving a new parameter with high
uncertainty, depends only on the estimated precision, regardless of the
estimated trueness 32.
Table 2. Intraday precision data for
ketoconazole in buffer pH 6.8
|
Concentration
standard (ppm)
|
Absorbance
|
Average±SD
|
RSD (%)
|
|
10
|
0.306
0.307
0.305
|
0.306±0.001
|
0.267
|
|
20
|
0.726
0.725
0.727
|
0.726±0.001
|
0.112
|
|
40
|
1.539
1.496
1.532
|
1.522±0.018
|
1.237
|
Table 3. Interday precision data for
ketoconazole in buffer pH 6.8
|
Concentration
standard 40 ppm
|
Absorbance
|
Average±SD
|
RSD(%)
|
|
Day-1
|
1.593
1.583
1.593
|
1.589±0.005
|
0.296
|
|
Day-2
|
1.648
1.648
1.651
|
1.649±0.001
|
0.008
|
|
Day-3
|
1.657
1.630
1.629
|
1.639±0.013
|
0.792
|
Limit of detection (LoD) and Limit of
quantitation (LoQ):
The LoD and LoQ
were determined to obtain the sensitivity of the developed method. LoD is the
lowest quantity of an analyte that can be detected, not necessarily determined,
and identified with a given degree of certainty 25,33. LoD is also defined as the lowest concentration that
can be distinguished from the background noise with a certain degree of
confidence. The result of LOD for ketoconazole was 0.988 that could be reliably
detected. LoD can be affected by minor changes in the analitycal system, e.g.
temperature, purity of reagents, matrix effects, instrumental conditions, but
LoD is not robustness parameter 33.
LoQ is the lowest concentration of an
analyte in a sample that may be determined with acceptable accuracy and
precision 25. The quantitation limit is a parameter of
quantitative assays for low levels of compounds in sample matrix and is used
particularly for the determination of impurities and/or degradation products 26. The result of LoQ for ketoconazole was 3.294 ppm
that represents the lowest concentration of the analytes that can be quantified
with acceptable accuracy and precision 34.
Approaches of LoD and LoQ may include
instrumental or non-instrumental procedures and could include those based on
visual evaluation, signal to noise ratio, standard deviation of the response
and the slope, standard deviation of the blank, and calibration curve 25. LoD and LoQ are sometimes useful to state a
concentration below which the analytical method cannot operate with an
acceptable precision 27.
Solubility determination:
The solubility of ketoconazole was
determined using buffer pH 6.8 after shaking using a shaker water bath. The
supernatant has been measured by a spectrophotometer at 230 nm (Table 5).
Table 4. Determination of sample concentration
|
Sample
|
Absorbance
|
Sample
concentration (ppm)
|
Average ± SD
|
RSD (%)
|
|
Replicate 1
|
0.844
|
21.569
|
21.044±0.759
|
3.61
|
|
Replicate 2
|
0.845
|
21.592
|
|
Replicate 3
|
0.773
|
19.971
|
The result of average was 21.044±0.759 ppm and % RSD
was 3.61%. However, the %RSD value still acceptable due to the requirements for
concentration analyte with 10 ppm is ≤7.3%, according to the AOAC
standard of the acceptable precision 31.
The solubility would be calculated to get Do use
equation 1 (Table 5) by relating oral dose and gastric volume (250 mL) to the
drug solubility in intestinal fluid 9, which was in this studied using buffer pH 6.8.
Dose 1
D0 = ------------ X
------------ (1)
250mL Solubility
Do > 1 predicts that the intestinal volume is
insufficient to dissolve the maximum dose, whereas Do < 1 predicts all drugs
will be in solution in intestines. In this study, the Do value was 38.066±1.4
that indicating ketoconazole was a poorly soluble drug in the intestine.
The solubility of ketoconazole in this study in
Phosphate Buffer Solution pH 6.8 is 21.044 ppm or 0.021044 mg/mL. In others
study, solubility ketoconazole have been determined in base pH solution to
predicted solubility of ketoconazole in intestine. Solubility ketoconazole in
human intestinal fluid is 0.029 mg/mL 35, in buffer pH 6.59 is 0.01 mg/mL 36, in FaSSIF is 0.0397 mg/mL. The result did not have
much difference in solution with pH around 6.
CONCLUSION:
Analysis of ketoconazole solubility in buffer
phosphate pH 6.8 has been using UV-Vis spectrophotometry with λmax
230 nm. Ketoconazole analysis method by UV-Vis spectrophotometry in this study
had linearity, accuracy, precision, detection limits, and quantization limits
that has been validated with accepted criteria. The proposed method is simple,
efficient without time-consuming sample preparation, and low cost. The method
can be applied for estimation concentration of ketoconazole in buffer 6.8
(alkali) solution.
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