The authors have declared that no competing interests exist.
The increasing use of
About 30 g of HF254 (Merck) silanized silica gel were suspended in 60 mL of a (2 : 1) water-methanol mixture by shaking vigorously for a few minutes in order to obtain a homogeneous suspension. The latter was then distributed on the plates using a Desaga spreader so as to obtain a uniform layer of 0.25 mm thickness. The plates were dried in air for about 3 h and stored over silica gel in a desiccator. They should not be used for at least 24 h after preparation.
Methanol solutions of pure commercial products (Merck, BDH, Carlo Erba) were used as reference solutions for
To identify the substances, the sample and reference solutions were applied as spots in parallel on the same plate using a micropipette and taking care to pipette no more than 5 µL each time, and to deposit from 2 to 10 µg of each preservative on the plate. For the quantitative determination, the solutions to be analysed were applied in bands on 5 x 20 cm plates, pipetting from 0.025 to 0.1 mL of solution so that from 20 to 100 µg of each substance are applied to each plate. The solutions were applied 2 cm from the starting edge leaving a margin of approx. 0.5 cm on both sides of the plate.
Chromatographic development was carried out using borate buffer, pH 2 (502 mL of 0.2 M H3BO3 solution diluted to 1000 mL with 0.2 M NaOH solution) as mobile phase, adding a given volume of organic solvent as required. The following buffer-solvent combinations have proved most suitable for separation of the various preservatives: buffer-methyl alcohol (90 : 10), buffer-ethylcellosolve (90 : 10), buffer-ethyl acetate (saturated solution at 25 OC), buffer-ethyl ether (saturated solution at 15 OC), buffer-tetrahydrofuran (95 : 5), buffer-dioxane (90 : 10).
The plates are developed in glass chromatography chambers lined with filter paper. Before putting the plates in them, the chrormatographic chambers were saturated with the solvent vapours for about 1 h. The plates are allowed to develop until the solvent front has risen about 14 cm from the point where the substance was deposited (90 to 120 min). When double development is desired, the plate is dried at room temperature for about 1 h and then returned to the chromatographic chamber. Chromatographic development is performed at about 25 OC except when the mixture contains ethyl ether in which case the temperature of the chromatographic chamber should be 15 OC.
After the plates have been developed they are dried at room temperature. The spots or bands of the various compounds were visualised under a 250-mµ UV light source.
After detection, the band of each substance is isolated by cutting away the sides of the layer of adsorbent with a spatula, and then performing the quantitative determination by removing the silica gel and extracting it with methyl alcohol. A silica gel collection method using a vacuum described by several authors
The eluent in the volumetric flask is diluted to volume with methyl alcohol and divided into two portions. One was acidified and the other was made alkaline by adding 2% of 1 N HCl and 1 N NaOH, respectively. Light absorption is measured at 297 mµ using the alkaline solution as test solution and the acidified solution as blank following the differential spectrophotometric method.
Compound | RF values |
|||||
M1 | M2 | M3 | M4 | M5 | M6 | |
Benzyl- |
0.04 | 0.08 | 0.03 | 0.02 | 0.03 | 0.08 |
Butyl- |
0.06 | 0.11 | 0.06 | 0.05 | 0.08 | 0.13 |
Propyl- |
0.15 | 0.22 | 0.14 | 0.15 | 0.14 | 0.20 |
Ethyl- |
0.23 | 0.32 | 0.25 | 0.25 | 0.24 | 0.28 |
Methyl- |
0.30 | 0.38 | 0.34 | 0.35 | 0.33 | 0.35 |
0.60 | 0.70 | 0.069 | 0.75 | 0.57 | 0.59 |
Each RF is the mean value of six separate chromatographic runs.
M1: Mixture A pH 2 buffer-methyl alcohol (90 : 10)
M2: Mixture B pH 2 buffer-ethyl cellosolve (90 : 10)
M3: Mixture C pH 2 buffer saturated with ethyl acetate at 25oC
M4: Mixture D pH 2 buffer saturated with ethyl ether at 15oC
M5: Mixture E pH 2 buffer-tetrahydrofuran (95 : 5)
M6: Mixture F pH 2 buffer-dioxane (90 : 10)
Of the mobile phases studied, mixtures C, D, E, and F gave the best separation of the various compounds, especially of benzyl- and butyl-
In quantitative zone chromatography, the separation may not be good enough for elution and quantitative determination of the components of the mixture when the amounts of some of the compounds to be separated are much larger than others. In this case, the mobility of the substances and the chromatographic separation can be greatly enhanced by performing a second development with the same solvent mixture, especially when low RF paraseptics are involved.
Compound | RF values |
|
M5 | M6 | |
Benzyl- |
0.06 | 0.17 |
Butyl- |
0.14 | 0.25 |
Propyl- |
0.24 | 0.38 |
Ethyl- |
0.36 | 0.49 |
Methyl- |
0.47 | 0.58 |
0.68 | 0.75 |
Each RF is the mean value of six separate chromatographic runs.
M5: Mixture E pH 2 buffer-tetrahydrofuran (95 : 5)
M6: Mixture F pH 2 buffer-dioxane (90 : 10)
The quantitative analysis of the eluents by differential spectrophotometry eliminates background absorption due to the extraction of the silica gel with methanol. The amount of the test compounds may be calculated from the spectrophotometric results using a calibration straight line plotted from results of chromatographic analyses performed on solutions containing known amounts of preservatives.
Greater precision in quantitative analyses may be obtained by using the internal standard method. In this case a known amount of a compound with a different RF from those of the test compounds is added to the test solution before chromatography (since the six preservatives discussed here are not generally used together in the same pharmaceutical speciality, one which is known to be absent in the test sample may be used as an internal standard). After chromatographic development and spectrophotometric determination of all the compounds, it is easy to calculate the amount of each single compound in the test solution using the formula:
Where, P is the amount of internal standard added, expressed in mg; EC and Est are the differential absorption of the eluents of the test compound and of the compound used as internal standard respectively; F is a correction factor calculated from the ratio between the differential absorptions of equal amounts of the internal standard and test compounds (experimentally, the F factor is determined by repeated quantitative chromatographic analyses of mixtures containing known amounts of the two compounds).
The data given in
Solution No. | Theoretical (mg/mL) | Found |
|||
Method A | Method B | ||||
mg/mL | % Difference | mg/mL | % Difference | ||
(a) Methyl- |
|||||
1 | 0.424 | 0.426±0.053 | +0.5 | 0.424±0.038 | 0.0 |
2 | 0.824 | 0.807±0.097 | -2.1 | 0.836±0.065 | +1.5 |
3 | 1.227 | 1.196±0.101 | -2.5 | 1.260±0.113 | +2.7 |
4 | 1.595 | 1.552±0.205 | -2.7 | 1.585±0.190 | -0.6 |
5 | 2.006 | 1.992±0.273 | -0.7 | 2.046±0.284 | +2.0 |
(b) Propyl- |
|||||
1 | 2.008 | 2.045±0.289 | +1.8 | 2.053±0.309 | +2.2 |
2 | 1.621 | 1.580±0.173 | -2.5 | 1.613±0.191 | -0.5 |
3 | 1.206 | 1.164±0.132 | -3.5 | 1.211±0.138 | +0.4 |
4 | 0.804 | 0.781±0.101 | -2.9 | 0.807±0.076 | +0.4 |
5 | 0.419 | 0.420±0.036 | +0.2 | 0.416±0.037 | -0.7 |
Mean of three chromatographic determinations performed under the same conditions.
Chromatography was performed on 5 x 20 cm H254 silanized silica gel plates using a pH 2 buffer-dioxane mixture (90 : 10) as mobile phase; 0.05 mL of sample solution was pipetted as a band on the plates. Method A: determination performed using the calibration straight line. Method B: determination performed by adding butyl-p-hydroxybenzoate as internal standard.
Current study | Bajaj and John |
|
Ion-exchanger | HF254 silanized silica gel | HF254 silanized silica gel |
Mobile phase | Borate buffer | Hexane: 1, 4 dioxane: methanol: triethylamine (7.5:1:1:0.5, v/v) |
Scanning densitometry at λmax | 250±1 nm | 254 |
Development distance | 12 cm | 1.5 cm |
Development time | 25 min | 30 min |
Concentration range | 8-150 μg/ml | Nd |
Equation | OD = -1.093 + 1.127 C | Nd |
r | 0.9996 | Nd |
Mean accuracy | 99.16±0.52% | Nd |
LOD | 0.24 µg per zone | 0.80 µg per zone |
LOQ | 0.43 µg per zone | Nd |
OD is the optical density (pixel intensity)
C is the concentration of Methyl-p-hydroxybenzoate
r is the regression coefficient
A repeatability test consisting of 15 chromatographic determinations on a methyl- and propyl-
The current method relies on the use of inexpensive equipment, a scanner and software, and does not use of critical derivatizing reagent, thus maximizing the ability of laboratories worldwide to analyze pharmaceutical samples. It also specifies a rapid separation of Benzyl-
Hydroxybenzoates are completely separated including a proper determination of