A simple and sensitive ultraviolet spectrophotometric method for quantitative estimation of glipizide in presence of lipid turbidity is described to avoid false estimation due to diffraction by turbidity. UV detection was performed at 230 nm, 225 nm, and 235 nm, and the calibration curve was plotted between resultant of absorbance of [230 nm − (225 nm + 235 nm)/2] and concentration of analyte. The calibration curve was linear over the concentration range tested (1–20
The samples of dissolution of liposomes/proliposomes contain lipid turbidity, as the literature review revealed that the drug released is measured after separation of the lipid turbidity in cooling ultracentrifuges at high speeds [
Derivative spectrophotometric methods, difference spectrophotometry, and bichromatic methods can also be applied for elimination of background absorption and/or scattering, but all these methods are more or less complex. Also derivative spectrophotometry results in more complex spectrum.
Various methods from different researchers are available for assay of GLIP. Some of them [
A simple, easy to calculate, ultraviolet spectrophotometric method is described here to eliminate the effect of lipid turbidity. The method is successfully applied to quantitatively estimate the drug in presence of lipid turbidity during dissolution as well as to determine drug content of the liposomes/proliposomal matrices. By this method the drug entrapped inside the liposomes/proliposomal matrices is not detected. Further, the same method is found to be applicable to quantitatively estimate the pure drug also. In the proposed method, there is no need to filter the samples to remove any undissolved drug before estimation because the undissolved drug in the samples does not absorb light, instead it diffracts.
In this method, the turbidity added from outside is in the form of blank processed lipid components of liposomes/proliposomes without drug in the appropriate ratio such that they form liposomes when come in contact with the aqueous medium. Now, as the lipid molecules self-assemble when they come in contact with the aqueous medium to make the lamellar bodies, the prepared turbid samples can be the proper model for real liposomal and proliposomal formulations.
All chemicals used were of reagent grade. Glipizide was kindly gifted by Alkem Laboratories Limited, Taloja, Raigarh, Maharashtra, India.
Acetonitrile (ACN), double-distilled water, sodium hydroxide, and potassium-dihydrogenorthophosphate were procured from Loba Chemie Pvt. Ltd., Mumbai, India.
A 1000
A 50
A 50
A Shimadzu Pharmspec UV 1800 ultraviolet-visible spectrophotometer was used.
Various aliquots of working stock solutions B and C were transferred to 10 mL volumetric flasks so as to prepare various alternate clear and turbid working standard dilutions of 1, 1.5, 2, 3, 4, 6, 8, 12, 16, and 20
The UV spectra of the prepared clear as well as turbid solutions of GLIP were run between 400 nm and 200 nm. Both exhibited a prominent peak at 230 nm (Figure
Overlain UV spectra of clear and turbid solutions of glipizide.
Standard curve.
The validity of the method for linearity, specificity, accuracy, repeatability, and precision according to recommendations was tested (ICH Guidelines Q2 (R1), 2005) [
Analytical parameters for determination of GLIP using the proposed method.
Parameter | Value |
---|---|
Analytical wavelengths (nm) | 225, 230, and 235 |
Equation used for standard curve |
|
Linearity range ( |
1–20 |
Regression equation |
|
Slope ( |
0.00533 |
Intercept ( |
0.00009 |
SD of intercept ( |
|
LOD (3.3 (SDintercept /Slope)) ( |
0.27 |
LOQ (10 (SDintercept/Slope)) ( |
0.82 |
Correlation coefficient | 0.99929 |
Test for residuals.
Predicted conc. ( |
Observed conc. ( |
Residual amount ( |
---|---|---|
1 | 1.202627 | 0.202627 |
1.5 | 1.571607 | 0.071607 |
2 | 2.057849 | 0.057849 |
3 | 2.9803 | −0.0197 |
4 | 4.010632 | 0.010632 |
6 | 5.852408 | −0.14759 |
8 | 7.777048 | −0.22295 |
12 | 11.99312 | −0.00688 |
16 | 15.77236 | −0.22764 |
20 | 20.32395 | 0.323952 |
Precision and accuracy of proposed method (
Solution |
Conc. analysed |
Intraday (Analyst 1) | Interday (Analyst 2) | ||||
---|---|---|---|---|---|---|---|
Found ± S.E.a,b |
Precision (%RSD) | Accuracy |
Found ± S.E.a,b |
Precision (%RSD) | Accuracy (%RME) | ||
Clear solution | 5 |
|
0.69 | 0.35 |
|
0.61 | 0.41 |
10 |
|
0.40 | 0.24 |
|
0.53 | 0.39 | |
15 |
|
0.52 | 0.32 |
|
0.44 | 0.30 | |
| |||||||
Turbid solution | 5 |
|
0.61 | 0.35 |
|
0.65 | 0.47 |
10 |
|
0.39 | 0.39 |
|
0.61 | 0.45 | |
15 |
|
0.50 | 0.43 |
|
0.61 | 0.39 |
bMean ± standard error.
%RSD: percentage relative standard deviation.
%RME: percentage relative mean error.
Recovery of the proposed method (standard addition method,
Dosage forms | Concentration taken, |
Concentration added, |
Proposed method %Recoverya,b ± %RSD |
---|---|---|---|
Pure GLIP | 10 | — |
|
8 |
|
||
10 |
|
||
12 |
|
||
| |||
Proliposomes of GLIP | 10 | — |
|
8 |
|
||
10 |
|
||
12 |
|
b%recovery =
Residual amount v/s variable curve.
The curve was found to be linear as shown by linear regression equation and the regression coefficient of the formed standard curve as shown in Table
To test the accuracy and precision of the proposed method a certain amount of GLIP was assayed by the proposed method three different times a day (intraday accuracy and precision) and three different days (interday accuracy and precision) in both clear and turbid solutions. The low values of %RME (represents accuracy) and %RSD (represents precision) indicate that the developed method is accurate as well as precise. Table
The repeatability and reproducibility of the given method are fairly good as indicated by the low values of %RSD and %RME.
As turbidity from outside was added to the alternate dilutions and the rest of validation parameters were within acceptable ranges, the method can be said to be specific.
The robustness of the method adopted is demonstrated by the consistency of the absorbance with the deliberate minor changes in the experiment, such as sonication time and pH of the buffer by ±1.
The recovery studies were performed by adding known amounts of the drug to the preanalysed samples from pure drug and in-house preparation at three different levels, that is, 80%, 100%, and 120% (standard addition method). The mean recoveries for GLIP were determined for both clear and turbid solutions (Table
The in-house formulation was added to a known sample and detected by the proposed method. It was found that the entrapped analyte was not detected by the proposed method.
The proposed method has been proved to be simple, precise, rapid, and reliable. The method was validated by evaluation of the validation parameters as described in the ICH2QR guideline [
Moreover, the method is fast with respect to analysis time as compared to sophisticated chromatographic techniques. No expensive instrumentation and no expensive organic solvents are required.
The method can be successfully employed for GLIP quantification in all types of pharmaceutical formulations and liquid samples of GLIP.
Authors are thankful to Alkem Laboratories Limited, Taloja, Raigarh, Maharashtra, India, for providing a gift sample of GLIP.