2 Susceptibility of surfactants towards pancreatic digestion
3.5 Results and Discussion
3.5.1 Analytical characterisation of the sucrose ester D1811
By means of HPTLC, SE D1811 could be separated into 10 fractions (Figure 26). According to Jasper et al., sucrose monoesters should have low migration distances, whereas diesters and higher esters migrate longer distances191. In order to verify the separation of the different esters, sucrose stearate products with an HLB range from 3 to 16 were spotted onto the HPTLC plate. The less polar SEs reflected by lower HLB values contain higher amounts of sucrose polyesters and less amounts of mono- or diesters. For polar SEs with high HLB values this effect was vice versa. For SEs with a HLB of 3, 5 or 7 the intensity of spots 8-9 was higher in comparison to the polar SEs with a HLB of 11 or 16. For the polar SEs with a HLB of 11 or 16, the intensity of spots 1-4 was much higher in comparison to the SEs with lower HLB values. The intensity of spots 5-7 was quite similar for all sucrose stearate types.
Figure 26 Photograph of a HPTLC plate after separation via a 4-step development program based on methanol/water/chloroform and post-chromatographic derivatisation.
Five sucrose ester (SE) preparations ranging from HLB 3 to 16 and stearic acid (SA) were spotted onto the silica plate representing 50 and 125 µg of the SE, respectively. The different SEs were separated into 10 fractions as illustrated in the drawing to the right. Less polar SEs reflected by lower HLB values contain higher amounts of sucrose polyesters and less amounts of mono- or diesters. For polar SEs with high HLB values this effect is vice versa. For SE with an HLB of 3 the intensity of spots 8-9 is higher in comparison to the polar SE with HLB 16. For the polar SE with HLB 16, the intensity of spots 1-4 is much higher in comparison to the SE with lower HLB values.
HLB 3 HLB 5 HLB 7 HLB 11 HLB 16 SA
1 2 3 4 6 7 10 9
5 8
Susceptibility of sucrose ester formulations towards pancreatic digestion Subsequent identification by MALDI-TOF mass spectra yielded into 4 peaks of monoesters (1-4) and 3 peaks of diesters (5-7) of sucrose and stearic as well as palmitic acid (Figure 26 and Table 17). Since there was no difference in the retention of stearic or palmitic derivatives, the presence of different peaks could be attributed to the different positions of esterification.
The lipophilic polyester suffered from poor ionisation. Peaks 8 and 9 could not be identified by MALDI-TOF-MS. According to Jaspers et al., these spots can be attributed to higher esters of stearic acid or palmitic acid with sucrose. Fraction 10 could be attributed to free fatty acids due to similar retention time of an external standard solution.
Table 17 Identification of the different fractions obtained after chromatographic separation of sucrose ester D1811 by correlation of the masses determined by MALDI-TOF-MS Peak Masses [Da] identification
1 603.4 631.4
[sucrose monostearate + Na]+ [sucrose monopalmitate + Na]+ 2 603.4
631.4
[sucrose monostearate + Na]+ [sucrose monopalmitate + Na]+ 3 603.4
631.4
[sucrose monostearate + Na]+ [sucrose monopalmitate + Na]+ 4 603.4
631.4
[sucrose monostearate + Na]+ [sucrose monopalmitate + Na]+
5 870.8
898.8 842.6
[sucrose palmitate-stearate + Na]+ [sucrose distearate + Na]+
[sucrose dipalmitate + Na]+
6 870.8
898.8 842.6
[sucrose palmitate-stearate + Na]+ [sucrose distearate + Na]+
[sucrose dipalmitate + Na]+
7 870.8
898.8 842.6
[sucrose palmitate-stearate + Na]+ [sucrose distearate + Na]+
[sucrose dipalmitate + Na]+
8 n.a. n.d.
9 n.a. n.d.
10 n.a. n.d. / fatty acid according to a reference solution
n.d. …not determined
Susceptibility of sucrose ester formulations towards pancreatic digestion
600 720 840 960 1080 1200
Mass (m/z) 10
40 70 100
%Intensity
631.8
603.8
870.2 898.3 647.8
600 720 840 960 1080 1200
Mass (m/z) 10
40 70 100
%Intensity
631.8
603.8
870.2 898.3 647.8
m/z: 603.8: [sucrose monopalmitate + Na]
m/z: 631.8: [sucrose monostearate + Na]
m/z: 647.8: [sucrose monostearate + K]
m/z: 870.2: [sucrose stearate-palmitate + Na]
m/z: 898.3: [sucrose distearate + Na]
+ + +
+ +
600 720 840 960 1080 1200
Mass (m/z) 10
40 70 100
%Intensity
869.8
897.8
841.7
600 720 840 960 1080 1200
Mass (m/z) 10
40 70 100
%Intensity
869.8
897.8
841.7
600 720 840 960 1080 1200
Mass (m/z) 10
40 70 100
%Intensity
603.3
631.4
600 720 840 960 1080 1200
Mass (m/z) 10
40 70 100
%Intensity
603.3
631.4
m/z: 603.3: [sucrose monopalmitate + Na]
m/z: 631.4: [sucrose monostearate + Na]
m/z: 869.8: [sucrose stearate-palmitate + Na]
m/z: 897.8: [sucrose distearate + Na]
+ +
+ +
725.3 725.3
Figure 27 MALDI-TOF mass spectra of sucrose ester D1811 (top) and exemplary spectra of isolated SE fractions 1 (bottom) and 5 (middle) after separation via HPTLC. Fraction 1 contains sucrose monoester and fraction 5 sucrose diester.
Further peaks which were detected from the isolated SE fractions could be attributed to matrix components deriving from the silica plate (e.g. peak at 725
Susceptibility of sucrose ester formulations towards pancreatic digestion 3.5.2 Susceptibility of sucrose ester D1811 towards lipolysis
In a first trial, the influence of enzymatic lipolysis on the SE composition should be monitored using the HPTLC method described earlier. Unfortunately, strong interferences between the hydrophilic components of the blank medium (phospholipids and bile salts) and the sucrose monoester fraction was observed (Figure 28). Hence, monitoring of changes in the SE compositions was not possible using this method. Therefore, the pH-stat titration had to be applied in the following experiments.
Figure 28 HPTLC chromatogram of blank medium (left) and sucrose ester D1811 (right) after separation utilising a 4-step gradient based on methanol/water/chloroform.
The components of the blank medium yield into many peaks interfering with the components of the sucrose esters. Hence, the monitoring of the in vitro digestion utilising this method was not possible.
As illustrated in Figure 29, the FA titration during incubation of the SE dispersion in simulated digestion media was in the range of the results obtained during hydrolysis of the blank medium. These results indicate that SE was not digested by pancreatic enzymes.
In order to evaluate the impact of the colloidal state of the SE, an emulsion of paraffin and SE D1811 was prepared and introduced to the digestion assay. The aqueous SE dispersions contain SE micelles. In the emulsion, SE stabilises the surface of a paraffin droplet. Paraffin is an indigestible oil which was used to avoid interferences of other lipolytic products. In Figure 29, it appears that higher FA concentrations were detected during simulated digestion of the SE-paraffin emulsion in comparison to the results of the blank digestion. The experimental error was in the range of 10%.
Susceptibility of sucrose ester formulations towards pancreatic digestion
0 15 30 45 60
0.0 0.5 1.0 1.5 2.0
0 50 100 150 200
FA titrated [µmol]
sucrose ester-paraffin emulsion sucrose ester dispersion Blank digestion
volume of 0.1 N NaOH [ml]
time [min]
Figure 29 Fatty acid concentrations as determined by pH-stat titration technique during a 60 min digestion period for mixed micelles (blank digestion), a sucrose ester D1811 dispersion (1% (w/v))) and a paraffin emulsion stabilised with 5% (w/v) of D1811 (data represent mean of 3 measurements). The release of FAs detected during simulated digestion of the SE substrate was in the range of the results obtained from blank experiments.
In a further experiment, it should be evaluated if the presence of SEs would have any influence on the digestion of a natural TG substrate for pancreatic lipase. For that purpose, the commercial fat emulsion Lipofundin® was diluted with varying concentrations of a SE dispersion before the lipolysis assay was started.
0 10 20 30 40 50 60
0 2 4 6 8 10
0 200 400 600 800 1000
FA titrated [µmol]
Lipofundin
Lipofundin + 0.5 % D1811 Lipofundin + 1.0 % D1811 Lipofundin + 2.5 % D1811 blank digestion
volume 0.1 N NaOH added [ml]
time [min]
Figure 30 Fatty acid concentrations as determined by pH-stat titration technique during a 60 min digestion period for mixed micelles (blank digestion) and the fat emulsion Lipofundin® which was mixed with different amounts of sucrose ester D1811 ranging from 0 to 2.5% (w/v (data represent mean±SD, n=3). All Lipofundin titration profiles were in the same range indicating that fat digestion
Susceptibility of sucrose ester formulations towards pancreatic digestion As illustrated in Figure 30, fat digestion was not influenced by the presence of the SEs under the experimental conditions. Neither a reduction of the FA concentration due to inhibitory effects nor increased FA levels due to a degradation of the SE itself were detected. This result further supports the thesis that SE D1811 was not susceptible to duodenal digestion.
In this study, sucrose ester D1811 did not undergo any detectable hydrolysis by pancreatic enzymes. These results were in contrast to Berry et al. who reported that the ester bonds of the sucrose ester mixture Sequol® 260 (containing 22% palmitic acid, 3.4% stearic acid, 22%
oleic acid and 47% linoleic acid derivatives) were cleaved by pancreatic juice and isolated pancreatic lipase to an extent of about 25-30% after 60 min188. Moreover, Berry et al.
concluded that the best hydrolytic activity was obtained with esters having a high content of unsaturated FAs. Nevertheless, they also observed that 15-20% of the esters of sucrose monostearate were cleaved by pancreatic lipase.
In contrast to these data but in agreement with the results obtained here, Christiansen et al.
recently reported that sucrose laurate (HLB 16) did not undergo any detectable hydrolysis by pancreatic enzymes using a similar digestion assay and FA titration as used in this study140. The reason for these oppositional findings remains unclear. Differences in the enzyme composition between the porcine pancreas extract preparation as used in this study as well as by Christiansen et al. and the enzyme preparations used by Berry et al. might be one explanation. It should be taken into consideration that the different methods used for the analytical evaluation of digestion hamper an adequate comparison of the results. The FA titration method for monitoring in vitro lipolysis might not be sensitive and precise enough to obtain discriminative results at FA liberations below 100 µmol.
Susceptibility of sucrose ester formulations towards pancreatic digestion
3.5.3 Dissolution of Ibuprofen sustained release tablets in biorelevant media
Following the investigations of aqueous SE formulations, a solid dosage form should be the focus of a further examination. It is well known that SEs can be used to modify the release of APIs. The mechanisms for this action are not yet completely understood. However, it has been discussed that the formation of H-bonds between the SE and the tablet filler causes the slow release of certain drugs186. Moreover, SEs form lyotropic mesophases upon contact with water, thus influencing the release of the drug186,187. In this section, dissolution testing as an established quality control method for oral solid dosage forms should be combined with the in vitro digestion assay. For that purpose, simulated digestion media were used as dissolution media.
During incubation in the different dissolution media, the Ibuprofen sustained release tablets underwent an erosion process: The size of the matrix tablets decreased in the course of the experiments (Figure 31). No initial swelling of the matrix system could be observed.
0 h 5 h
7 h 12 h
Figure 31 Photographs taken during dissolution of ibuprofen sustained release tablets
‘Ibuprofen 800 AL retard’ in phosphate buffer (paddle apparatus, pH 6.8).
Susceptibility of sucrose ester formulations towards pancreatic digestion Figure 32 shows the mean dissolution profiles of Ibuprofen sustained release tablets in various dissolution media. The dissolution profile in phosphate buffer showed a slow, non-linear drug release within 12 h. Half of the Ibuprofen content was released after about 3.8 h.
The dissolution profile of Ibuprofen in buffer containing additional calcium and sodium chloride was slightly faster. 50% of the API was released after about 3.2 h and dissolution was already completed after 10 h. The dissolution of Ibuprofen in bioreleavant media containing bile salts and phospholipids was considerably faster compared to the dissolution in buffer. 50% of the API was released after about 2-2.2 h and dissolution was already completed after 8 h. Surprisingly, no difference in the dissolution profile was observed if low amounts of BS/PL reflecting fasted state intestinal conditions or high amounts of BS/PL reflecting fed state intestinal conditions were used. Furthermore, pancreatic enzymes which were present in the media reflecting fed state conditions had no influence on the dissolution of the API from the matrix tablets.
0 2 4 6 8 10 12
0 20 40 60 80 100
Dissolution in buffer
Dissolution in buffer with salts Dissolution in FaSSIF
Dissolution in FeSSIF with Pancreatin
Ibuprofen released [%]
time [min]
Figure 32 Mean dissolution profile of Ibuprofen sustained release tablets ‘Ibuprofen 800 AL retard’ in phosphate buffer and biorelevant media (FaSSIF and FeSSIF with pancreatin). Experiments were run in triplicate using USP dissolution apparatus 2 (paddle) at a rotation speed of 75 rpm.
The faster dissolution of the Ibuprofen tablets in buffer containing additional salts in comparison to the native buffer can be attributed to the higher ion strength of the dissolution medium. By inclusion of bile salts and phospholipids, the wettability of the solid dosage form was improved. Furthermore, it is well known that the saturation solubility of a drug can be substantially increased by the presence of mixed micelles42. These aspects contribute to a higher dissolution rate of Ibuprofen in biorelevant media. In accordance with the previous results obtained by in vitro digestion experiments with sucrose stearate, no difference in the
Susceptibility of sucrose ester formulations towards pancreatic digestion These results further underline the thesis that SEs of higher saturated FAs are poor substrates for pancreatic lipase.