Fat emulsions for parenteral nutrition II: Characterisation and physical long-term stability of Lipofundin MCTLCT

Chid

Nutrition (1993) 12: 29&309 @ Longman Group UK Ltd 1993

Fat emulsions for parenteral nutrition II: Characterisation and physical long-term stability Lipofundin MCT/LCT

of

R. H. MLJLLER and S. HEINEMANN Department of Pharmaceutics and Biopharmaceutics, Kiel, Germany (Correspondence and reprint requests

University to RHMl

of Kiel, Gutenbergstr.

76-78, D-24 118

ABSTRACT-Lipofundin MCT/LCT* emulsions for parenteral nutrition were characterized in terms of particle size (bulk population), presence of large particles (>> 1 urn) and droplet charge (zeta potential as measure of electrostatic stabilization). Photon Correlation Spectroscopy (PCS), microscopy and Laser Diffractometry (LD) were employed as sizing techniques and compared to each other. Zeta potentials were determined by Laser Doppler Anemometry (LDA). The characterization data showed a good batch-to-batch reproducibility. The physical stability of Lipofundin MCT/LCT was monitored over a period of 2 years. The mean diameter of the bulk droplet population did not increase. No formation of larger droplets was found by microscopy and LD. A negligible coalescence could only be detected by the very sensitive PCS measurements (two time window analysis). The zeta potential stayed above -45 mV providing sufficient electrostatic stabilization during the whole storage period.

Introduction Size of the bulk population, percentage of particles larger than the bulk population, maximum particle size and charge of fat emulsions are important characterization parameters of fat emulsion for parenteral nutrition. Changes in these parameters during storage are a measure of physical stability. The magnitude of the droplet charge (zeta potential) gives an indication of the contribution of the electrostatic repulsion to emulsion stability (1). The in vivo fate and organ distribution depends also on size and charge. Large and/ or highly charged emulsion droplets are cleared more rapidly from the circulation by the macrophages of the liver (2, 3). Large droplets or flocculated emulsions are localized in the lungs by a process of mechanical filtration (4). Massive embolization of the capillary bed can lead to physiological (5) and haemodynamic effects (6). Increased toxicity with increasing droplet size has also been reported for fat emulsions (7-9). Accelerated tests are very often employed to predict the stability of emulsions (10-12). Such applied stresses however change the properties of the emulsions (e.g. reduction in microviscosity (13) at higher

* in Germany:

Lipofundin”

MCT, B. Braun Melsungen

A G

temperatures). Long-term stability tests are more time consuming, but avoid such changes. Much attention has been focussed on the stability of mixed nutrition solutions (12, 14); less data has been published about particle size and charge changes in the emulsions themselves. The aim of this paper was therefore to study alterations in a fat emulsion under normal storage conditions over a long-period. A range of previously described and evaluated methods were used to obtain as complete a picture as possible of the physical stability of a new generation fat emulsion, Lipofundin MCT, which contains 50% medium chain triglycerides (MCT) in the oil phase (16-24). Over a long period, the properties of the emulsifier film may be affected by interaction with the oil and displacement of LCT by MCT. This produces no effect on interfacial tension (25) but a slight decrease in the viscosity of the emulsifier film (26), described as ‘an effect of external environment’ (13). This study was designed to examine the effect of these changes on long-term stability. Materials Samples of four batches of Lipofundin MCT/LCT emulsion were purchased from B. Braun Melsungen 298

CLINICAL NUTRITION

AG (Germany) containing 10% (batch A: batch no. 551281A; batch B: 604581A) and 20% fat (batch A: 604582A; batch B: 606182A). The composition of the 10% emulsions was: 50.0 g soya oil, 50.0 g mediumchain triglycerides (MCT), 12.0 g 3-sn-phosphatidyl choline (egg lecithin), 25.0 glycerol and water added to 1000 ml. The 20% emulsions contained 100 g of soya oil and MCT respectively but identical amounts of lecithin and glycerol (Trade names: Lipofundin MCT (Europe), Medialipid (France), Vasolipid (Norway)).

Methods Photon Correlation Spectroscopy was used to determine the mean particle size of the bulk population (PCS diameter) and the width of the size distribution (polydispersity index, PI) (15). A two-time window analysis as described previously (15) was applied to assess the number of particles present, which are distinctly larger than the main population. A short sample time (25 ps) and a long sample time (100 us) were used in the PCS measurement. The first shows the diameter of the bulk population (D25 ps), and the second will shift to larger values with an increase in the number of larger particles. The difference between the two diameters (AD = DlOO 1s - D25 us) is therefore a measure of the fraction of larger particles present in the emulsion. Alternatively, the sample time can be adjusted to obtain a decay of the measured correlation function to l/e at a constant channel, e.g. 20. This diameter however depends very much on the fraction of larger particles present rather than the diameter of the bulk population. It is the method used in the software routine of autosizers to find the sample time. The PCS system consisted of a Malvem Spectrometer RR102 connected to a 4-bit correlator K7025 and a logarithmic correlator K7027 (Malvem). The logarithmic correlation functions were transformed to a size distribution as described previously (15). A Laser Diffractometer (2600, Malvem Instruments, UK) was used to determine the particle size between 0.5 pm and 100 urn. To characterize the distribution, the weight diameters D(50%) and D(90%) and the volume mean diameter VMD were calculated. The diameter D(90%) is most sensitive towards large particles and will show an alteration accordingly. In addition the upper size limit of the largest size class is given, in which particles were detected (15), e.g. 100% of the particles are ~2.4 p.m. A Coulter Counter model TA II was used for single particle counting. The emulsion was dispersed in Isoton II (0.9% NaCl solution, Coulter Electronics,

299

UK); measurements were performed with a 30 pm capillary. Three samples were drawn per batch and measured three times each. Channel 1 (0.50-0.63 pm) of the Coulter Counter was switched off because too many particles were counted in this channel (up to 10 times the number as in channel II; this would lead to an automatic reset in channel I above 99 999 particles). The measurement was terminated by a preset count number of 90 000 particles in channel 2 (0.630.79) urn). The counts of the 9 measurements per batch were added up to a total which is given in the tables. Light microscopy was used to assess the quality of the emulsions using a scheme described previously (15). Microscopic photographs were taken with a micro flash system (Supra, FRG) and examined with regard to the presence of larger particles. Zeta potential (ZP) measurements were performed by electrophoretic measurements using a Malvem Zetasizer II (Malvem Instruments, UK). Measurements were performed in distilled water at pH 5.3 and in phosphate buffer (Sorensen pH 7.4,O.Ol M), 4 mm glass capillary, applied field strength 20 V/cm. The electrophoretic mobility (EM) was measured by Laser Doppler Anemometry (LDA) and transferred to a zeta potential by the Helmholtz equation (15). For measurements in water at 25°C the equation can be simplified to: ZP (mV) = 12. 8 EM ((u/cm)/(V/cm)). Sample numbers: 3 bottles of each batch were investigated after each time interval. Photon Correlation Spectroscopy: 1 sample was drawn per bottle (10 measurements per sample) and a mean diameter of each bottle calculated. From the mean diameters of the bottle a mean diameter of the batch was obtained. Laser Diffractometry (LD): 3 samples per bottle were taken (3 measurements per sample), mean values were calculated from the total of 9 measurements. The LD results were very consistent, in general 3 identical readings were obtained with regard to maximum particle size detected and the various mean diameters. Microscopic analysis: 3 samples were drawn from each batch to take micrographs. Zeta potential measurements: 1 sample was taken per bottle (5 measurements per sample). A mean zeta potential of each bottle was calculated and from these data a mean zeta potential of the batch was obtained.

Results and discussion Sire and width of bulk populations

(PCS)

The mean PCS diameters (D25 us) of the two batches of 10% emulsions were both in the range of 265-

300 FAT EMULSIONS FOR PARENTERAL NUTRITION II

Table 1 PCS data of Lipofundin MCT/LCT 10% emulsions determined over a period of 24 months (D25 us -mean diameter of bulk population; DlOO us - diameter weighted by larger particles; AD = DlOO ps - D25 us; PI polydispersity index) Time (months) 0

6 12 18 24

Lipofundin MCT/LCT D25 us DlOOus 213 271 274 272 273

nm nm nm nm nm

284 284 291 290 291

nm nm nm nm nm

10% batch A AD PI 11 nm 13 nm 17 nm 18 nm 18 nm

0.119 0.088 0.104 0.103 0.104

Lipofundin D25 us

MCT/LCT DlOOus

264nm 262 nm 265 nm 263 nm 266 nm

278 272 282 283 284

nm nm nm nm mn

10% batch B AD

PI

14 nm 10 mn 17 nm 20 nm 18nm

0.119 0.096 0.098 0.098 0.109

Table 2 PCS data of Lipofundin MCT/LCT 20% emulsions determined over a period of 24 months (D25 us -mean diameter of bulk population; DlOO us - D25 us; PI polydispersity index) Time (months) 0 6 12 18 24

DlOO its -diameter

Lipofundin MCT/LCT 20% batch A DlOOys AD D25 us PI

Lipofundm D25 ps

332 nm 321 nm 320 nm 319nm 323 nm

365 nm 374mn 370nm 372nm 372 nm

348 337 343 346 352

nm nm nm nm nm

16nm 16nm 23 nm 27 nm 299 nm

0.136 0.136 0.126 0.130 0.143

275 nm. The polydispersity indices were about 0.100 (Table 1). This indicates a relatively narrow distribution when considering the PI of ‘monodisperse’ latex standard particles in the range of 0.030-0.070. There was good reproducibility of both the mean droplet size and the narrow width of the bulk population. The consistency between the batches was high. The mean PCS diameters of the 20% fat emulsions were distinctly higher (330 mn-370 nm) and different between the two batches (Table 2). The increase in the mean droplet size can be explained by a decrease of the dissipation Pv (power density) during the high pressure homogenization. The dissipation decreases with increasing concentration of the dispersed phase (oil) leading to an increase in the droplet diameter (27). The increase in the concentration of the dispersed phase could also affect the distribution of the dissipation throughout the homogenization zone (power density distribution). The distribution needs to be homogenous to produce uniform droplets (27). A heterogeneity of the distribution of the dissipation can therefore explain the increased polydispersity index obtained for the 20% emulsions (0.140-o. 180). The slight variations in diameter and polydispersity between the batches are also attributed to the less controllable heterogenous distribution of the dissipation (Table 2). For Intralipid 10% a mean PCS diameter of 285 run and a polydispersity index of 0.236 have been reported (28). This would indicate a distinctly

weighted by larger particles; AD =

MCT/LCT 20% batch B DlCKtps AD 393 398 400 403 403

nm nm nm nm nm

25 24 30 31 31

nm nm nm nm nm

PI 0.212 0.180 0.181 0.183 0.184

broader size distribution. In comparative measurements with Intralipid lo%, mean diameters of about 280 nm and a polydispersity between 0.130-0.150 were obtained (unpublished data). The low standard deviation of the PCS measurements and the consistency of the mean diameters between bottles within one batch demonstrated the reproducibility of the PCS. Low relative standard deviations of about OS-2% (1.5 mrt-5 nm) allowed the reproducable PCS results to be obtained during the storage period of 2 years. The mean diameters of the bulk populations did not change for all four batches (Tables 1 & 2). The variation widths of the measured diameters over 24 months were 3 nm and 12 nm for the 10% batches, 13 nm and 10 nm for the 20% batches. The polydispersity indices were also consistent during the storage period considering the standard deviation in the range of 5-15%. From these PI data no droplet coalescence could be detected. Presence of particles larger than the bulk population (PCS). The magnitude of D was shown to be correlated to the amount of particles present, which are distinctly larger than the bulk population. The two 10% emulsions possessed values of 11 run and 18 nm (batch A, B), and the 20% emulsion of 16 nm (A) and 25 nm (B). This parameter allows differentiation between the

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301

Table 3 Size differences AD related to the microscopic assessment of emulsion quality. The microscopic scheme is based on the diameter of the bulk population and the extent of larger particles present. The four emulsions (A-D) were especially produced test emulsions with increasing numbers of large particles ( 1) Emulsion

A B C D

AD size difference (nm)

16 40 64 383

mean droplet size of bulk population (mn)
size of larger oil droplets (pm) which are present to a large extent

extent

1.0-1.5 1.o-2.0 1.5-2.5 2-5

2.0-3.0 2.5-3.0 5-8

emulsions. The 10% emulsions and batch A of the 20% emulsions contain the least number of larger particles. To relate the AD data to a quality assessment we refer to the scheme published previously (15). (Table 3). Values of about 16 nm are obtained in ‘very fine’ emulsions, which contain a very low number of larger particles. The size range of these largest particles present is 2-5 l,trn. From the correlation between AD and the microscopic evaluation scheme all of the batches can be assessed as ‘very fine’ emulsions. The polydispersity index has a relatively large standard deviation (5~15%). The AD values are calculated from the two mean diameters D25 ps and DlOO ps which possess a distinctly lower standard deviation of 0.5-2.0%. Therefore they are more sensitive than the PI in detecting an increase in the number of larger particles. The AD values increased from 11-18 run for batch A(lO%) indicating negligible coalescence. The AD of batch B(lO%) was in the range of 17-l 8 nm over the whole storage period. The AD values of the 20% batches showed a slight increase from 16 rim/25 nm to about 30 nm. With a AD = 18 nm, after 2 years of storage, the 10% emulsions will be still in the category of ‘very fine’ emulsions. Despite the slight increase in AD to 30 nm, the 20% emulsions are between the categories ‘very fine’ and ‘fine’ (c. f. Table 3). Size distribution of emulsions (PCS polydispersity analysis) The correlation functions were transferred to a size distribution by fitting 5 delta functions using a low resolution (Figs 1 & 2). The low resolution leads to relatively broad distributions which show the presence of a small weight fraction or larger particles up to l-2 pm. However, the width of the bulk population appears to be broadened. Therefore two delta functions were fitted and the resolution was increased.

IOW

1.5-2.0

emulsion assessment

low extent but detectable

very

2-5 3-6 5-8 >lO

very fine fine medium coarse

This led to a more realistic width of the bulk population but the larger particles could not be detected any more (Figs 1 & 2), since they disappeared in the noise of the transform. To characterize the emulsions, an analysis by both 5 and 2 delta functions with low and high resolution is required. The fitted size distributions were found unsuitable for detecting differences between very similar emulsions (e.g. batches of 10% emulsions). The emulsions need to be different as for example B(lO%) and B(20%). For this reason only the transforms for these two emulsions are shown (Figs 1 & 2). To detect time-dependent alterations the transforms at 0 months and 24 months were compared. The distributions obtained by fitting 5 delta functions show even fewer particles above 1 pm (Figs 1 & 2). Therefore the method seems unsuitable for detecting small increases in the number of larger particles. Calculating AD values appeared to be much more sensitive. The distributions obtained by fitting two delta functions are very similar at 0 and 24 months. The shape of the histograms changed very little. The method does not appear sensitive enough to draw any conclusions with regard to changes in the distribution of the bulk population - despite the fact that the transform conditions were kept constant (number of delta functions, resolution). Automatic apparatuses calculate the distributions with varying transforms conditions. The width of the distributions will mainly be influenced by the number of delta functions and the resolution applied, and less by true differences between emulsions (15). The influence of these parameters is obvious in Figures 1 & 2. The value of automatically calculated distributions to compare emulsions is therefore questionable. Presence of larger particles (Laser Diffractometer) Laser Diffractometer analysis covers the droplet size range above 0.5 pm. The bulk populations of

302 FAT EMULSIONS FOR PARENTERAL NUTRITION II 0 months

N

0 months

24 months

N

24 months

1

weight

distribution.

5 delta

functions

weight

0 months

24 months

20

50

weight

100

distribution,

200

2 delta

5bo

distributlan,

5 delta

functions

0 months

N

24 month.3

“I

1000 (nm)

functions

weight

distribution,

2 delta

funCtiOnS

Fig. 1 Size distributions of Lipofundin MCT/LCT 10% (batch B) obtained by fitting 5 delta functions at a low resolution (upper 2) and by fitting 2 delta functions at a high resolution (lower 2). 0 and 24 months storage time respectively.

Fig. 2 Size distributions of Lipofundin MCT/LCT 20% (batch B) obtained by fitting 5 delta functions at a low resolution (upper 2) and by fitting 2 delta functions at a high resolution (lower 2). 0 and 24 months storage time respectively.

270 mn-370 nm cannot be detected and are not considered when calculating the diameters and the percentage of particles in different size classes. The diameter D50% is therefore the mean of the particles between 0.5 pm and 5 pm and consequently higher than the PCS mean diameter. No differences in the diameters 50% and 90% and the volume diameter were detected between the batches of emulsions with identical oil concentration (Table 4). These data confirm the batch consistency observed above. However, the diameters for the 20% emulsions are slightly higher than the ones determined in 10% emulsions. This corresponds to the maximum particle size detected. All particles were below 2.4 pm in the 10% emulsions, below 3.9 pm in the 20% emulsions. The presence of slightly larger

particles in the 20% emulsions can be attributed to the different dissipation distribution. The size distributions of the droplets above 0.5 pm are given in Table 5. About 97% (10% emulsion) and 90% (20% emulsions) of the particles are in the size class 0.5-1.2 pm. The particles detected by the LD are only a small fraction compared to the bulk population. The droplets above 1.2 pm are therefore an extremely small fraction of the total particle number. Considering the portion and size of the largest particles detected, the fat emulsions will not cause any toxic effects due to capillary blocking. To assess the quality based on LD data, the diameters and the maximum particle sizes of the emulsions (Table 4) were compared with the data of the four standard emulsions (Table 6). The 10% emulsions are

CLINICALNUTRITION

Table 4

Laser Diffractometer data of the Lipofundin MCT/LCT emulsions over a storage period of 24 months (D(50%), D(90%) -diameters 50%/90%, VMD volume mean diameter, 100% < - 100% of the particles are below the stated size) Time 0

12 24 0

12 24 0

12 24 0

12 24

D(50%)

D(90%)

VMD

100% <

10% batch A batch no. 551281A

0.78 pm 0.78 urn 0.78 urn

1.16 pm 1.16 urn 1.15 urn

0.79 urn 0.79 urn 0.78 urn

2.4 urn 3.9 pm 2.4 pm

10% batch B batch no. 604581A

0.77 urn 0.78 urn 0.79 urn

1.16 urn 1.15 urn 1.16 urn

0.79 pm 0.79 pm 0.80 urn

2.4 urn 3.9 urn 3.9 urn

20% batch A batch no. 604582A

0.82 pm 0.81 urn 0.82 urn

1.22 urn 1.22 urn 1.23 pm

0.84 pm 0.84 urn 0.84 urn

3.9 urn 5.0 urn 3.9 urn

20% batch B batch no. 606182A

0.82 ym 0.82 pm 0.82 mm

1.26 urn 1.25 urn 1.25 mm

0.86 urn 0.86 urn 0.85 mm

3.9 urn 5.0 pm 3.9 mm

Lipotimdin

MCT/LCT

Table 5 Size distribution of the larger particles in Lipofundin MCT/LCT 10% and 20% over a storage period of 24 months: percentage of particles (fraction) in different size classes. The data do not consider emulsion droplets below 0.5 pm

Time

Lipofundin MCT/LCT

0.5-l .2

1.2-1.5

fraction (%) in size range 1.5-1.9 1.9-2.4 2.4-3.0

3.0-3.9

3.9-5.0

0 12 24

10% batch A 551281A

91.2 97.0 97.9

1.43 1.56 1.04

1.06 0.85 0.78

0.34 0.36 0.25

_

_

_

0.12 _

0.17 _

_

0 12 24

10% batch B 604581A

96.8 97.9 96.6

2.00 0.98 2.10

0.10 0.71 0.95

0.27 0.31 0.26

0.02 0.09 0.03

0.01 0.01

_ _ _

0

20% batch A 604582A

90.2 90.6 90.0

7.17 6.91 7.16

2.25 1.91 2.22

0.34

0.06

0.02

_

0.40 0.48

0.22 0.14

0.07 0.05

0.08 _

20% batch B 606182A

89.2 89.3 89.2

7.25 7.19 7.23

2.29 2.27 2.30

0.82

0.30

0.10

_

0.80 0.80

0.30 0.29

0.12 0.10

0.08 _

12 24 0

12 24

Table 6 Relation between PCS size difference AD, laser diffractometer data and microscopic assessment of emulsion quality. The microscopic scheme is based on the diameter of the bulk population and the extent of larger particles present (c.f. Table 3) Standard

Size

emulsion

difference (mn)

A B C D

16 40 64 383

Laser Diffractometer D(50%)

D(90%)

0.77 0.82 0.93 1.88

1.13 1.22 1.72 3.47

pm pm urn urn

pm pm urn urn

data

VMD

0.77 0.84 1.00 2.01

urn urn urn urn

100% <

1.2 3.9 3.0 6.4

pm pm pm j.tm

microscopic emulsion assessment very fine fine medium coarse

303

304 FAT EMULSIONS FOR PARENTERAL NUTRITION II

identical to standard emulsion A(very fine), the 20% emulsions are identical to standard emulsion B (fine). The laser diffractometer diameters stayed unchanged during the storage period. The maximum particle sizes detected were between 3.9 pm-5.0 pm. The emulsions maintained therefore their high quality categories of ‘very fine’ and ‘fine’ throughout the whole storage period of 2 years.

Microscopic assessment of emulsions The microscopic photographs showed that the mean droplet diameters of the bulk populations are below 1 pm for all the emulsions (Figs 3 & 4). Droplets up to 1.5 pm are present to a large extent, particles between 1.5-2.0 pm to a low extent. Particles in the range 2-5 pm are very few but still detectable (20% emulsion: up to 6 pm). The microscopy is in agreement with the laser light scattering characterization data. As a single particle counting technique, it is able to detect a few larger particles which are not detectable by LD. This explains the slightly higher values for the maximum particle size detected (15). The microscopic photographs taken after 2 years of storage show no difference to the ones at 0 months (Figs 3 & 4, lower). No formation of larger droplets was detectable by microscopy. This is again in agreement with the LD stability data. The slight increase in AD measured in the 20% emulsions corresponds to a minor coalescence below the detection limit of the other methods. In turn, this demonstrates the sensitivity of the PCS two time window analysis for monitoring changes in emulsions.

Coder Counter analysis of the emulsions At 0 months the fat emulsions were analysed with a Coulter Counter to enable a comparison to LD and microscopic assessment with regard to the presence of large particles. Particles up to 4 pm (batch A) and 5 pm (batch B) were detected in the 10% fat emulsions, in some of the nine measurements of each batch a few single particles between 5 pm and 8 pm (Table 7). Relative to the number in the lowest size class in which particles were counted (810 000 between 0.63-0.79 pm) this fraction is extremely low (4 particles). This fraction is even lower considering that the particle number in the lowest size class (0.500.63 ym) was up to ten times the number in the class 0.63-0.79 nm. The particles above 5 pm were detected because it is a single counting technique. However, they were few and may have been artefacts. They are therefore not considered in the comparison between the techniques.

As found previously (15) the Coulter Counter is slightly more sensitive in detecting a few large particles than the LD. In batch A( 10%) and B( 10%) particles in the size classes up to 4.00 l.tm/5.04 pm were detected in contrast to 2.4 pm/3.9 pm by the LD. The maximum particle size was also slightly larger for the 20% emulsions (5.04/6.35 pm compared to 3.9 pm by the LD). However it should be pointed out that these larger maximum sizes detected by the Coulter Counter are due to only 4 particles in a population of >> 810 000. The upper maximum sizes measured by LD and Coulter Counter are therefore regarded as being identical. The LD measurements are less timeconsuming and were therefore used to monitor the stability over 24 months. The particles below 0.5 pm are outside the measuring range of the Coulter Counter and the LD. The particle size distributions measured do not include the droplets of the main populations below 0.5 pm. The microscopic technique provides some estimation of the relative fractions of the larger particles to the main population at about 0.3 pm. It was therefore chosen as second technique to monitor long-term stability. In addition, the microscope was found to be most sensitive in detecting a few larger particles (15).

Zeta potential measurements The zeta potentials in distilled water were identical for the 4 batches of emulsions differing in the mean particle size but produced with the same chemical compounds. The zeta potential does not depend on the size for small particles with a large diffuse layer (29). The diffuse layer is large due to the low electrolyte concentration in the fat emulsions. The zeta potentials were therefore expected to be similar or identical. The measurements were performed in distilled water to avoid a change of the potential due to adsorption of electrolytes or compression of the diffuse layer (3s 32). Measurements in 0.01 M phosphate buffer led to distinct ZP reductions for some of the emulsions (ZP = -25--30 mV); others were less affected. For Intralipid a ZP of -3 1.5 mV in 0.01 M HEPES buffer has been reported (28). However, this low value does not reflect the droplet potential in the electrolyte-free emulsion. To keep the results comparable and to imitate the electrolyte concentrations in the infusion bottle, ZP determinations were performed in distilled water at pH 5.2 (Table 8). The ZP values of 46--49 mV are in the range providing moderate electrostatic stabilization of suspensions (-31--60 mV). The ZP should be -61-80 mV for good and between -81 and -100 mV for excellent stabilization (1). Aggregation in suspensions

CLINICALNUTRITION

Fig. 3 Microscopic photographs of the batches A & B of Lipofundin months, upper) and after 24 months of storage (lower). Bar: 5 pm.

MCT/LCT

10% (left: A; right: B) after preparation

(= 0

305

306

FAT EMULSIONS FOR PARENTERAL NUTRITION II

Fig. 4 Microscopic photographs of the batches A & B of Lipofundin MCT/LCT 20% (left: batch A, right: batch B) after preparation (= 0 months, upper) and after 24 months of storage (lower). Bar: 5 pm.

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307

Table 7

Coulter Counter analysis of the Lipofundin MCT/LCT emulsions at 0 months. Three samples were drawn per batch, each sample was measured 3 times, the measurement was stopped when 90 000 particle counts were obtained in channel 2 (size class 0.630.79 pm, 9 measurement x 90 000 particles = 810 000 particles). The particle number in the size class 0.5m.63 pm is up to 10 times the number in the class 0.63-0.79 pm Size class (pm)

batch A (10%)

0.50-0.63 0.63-0.79 0.79-l .oo 1.00-1.26 1.2c1.59 1.59-2.00 2.00-2.52 2.52-3.17 3.17~.00 4.00-5.04 5.04-6.35 6.35-8.00 > 8.00

Table 8 Time (months) 0

12 24 36*

0 12 24 36*

particle number counted in the size classes for batch B (20%) batch B (10%) batch A (20%)

810000 50 039 1348 251 50 10 14

_

_

810000 78 049 1464 380 92 25 11 4

810 000 108 756 3305 514 137 53 17 6 4 0 2 0

0

3 0

Zeta potentials of Lipofundin

MCT/LCT

10% and 20% in distilled water over a period of 36 months

Lipofundin

MCT/LCT 10% batch A 551281A zeta standard deviation potential

-48.7 -54.8 -58.1 -47.7

-46.4 -51.1 -58.8 -47.9

810000 136 394 8750 958 205 63 14 12 2 2 0 0

mV mV mV mV

0.82 mV 0.5 1 mV 0.70 mV 0.43 mV

Lipofundin

MCT 20% batch A 604582A

mV mV mV mV

0.56 0.58 1.10 0.42

Lipofundin

mV mV mV mV

MCT/LCT

zeta potential 46.1 -51.8 -62.0 46.2

mV mV mV mV

10% batch B 604581A standard deviation 0.69 0.58 0.60 0.38

mV mV mV mV

Lipofundin

MCT 20% batch B 606182A

-46.6 48.0 -60.2 118.4

0.74 0.29 1.10 0.46

mV mV mV mV

mV mV mV mV

(* The 36 months data are taken from (34)).

is promoted due to the reduction of the interfacial area, van der Waals attraction and hydrophobic interactions between lipophilic particles. The fat emulsions proved to be stable despite the relatively low zeta potentials. This might be explained by a low hydrophobic interaction between emulsion droplets. The hydrated emulsifier film was found to be relatively hydrophilic as determined by hydrophobic interaction chromatography (I-UC) (11, 33). The stability of the emulsifier film appears therefore to be an important factor in preventing coalescence. The rigidity depends on the microviscosity of the phospholipid film (34). The contribution of the film rigidity compensates for the missing electrostatic repulsion, and the emulsions are therefore stable despite a zeta potential below the value required for good electrostatic stabilization. Reduction of one of the

complementary forces leads to droplets coalescence (e.g. reduction of the electrostatic repulsion in TPN regimens with high electrolyte load, reduction of microviscosity in emulsions containing amphiphilic drugs). During the storage period of 24 months the zeta potential stayed above 45 mV and there was no reduction in the electrostatic stabilization of the emulsions. The zeta potentials were slightly increased at 24 months of storage. Washington et al (35) attributed such an increase to chemical decomposition of the lecithin. The formation of lysolecithin and free fatty acids can increase the droplet charge. Indeed, zeta potentials, up to -90 mV can be obtained by the penetration of negatively charged compounds into the lecithin layer (e.g. sodium lauryl sulphate (36)). However, the increase in the potential at 24 months was

308 FATEMULSlONSFORPARENTERALNUTRITIONIl

attributed to a slightly higher electrolyte concentration of the distilled water. To avoid an interfering pH effect the pH of the distilled water needed to be adjusted to 5.2 by addition of NaOH. The interference by the added electrolyte was less than the disturbance caused by a low pH. Slight increases in the conductivity raise the potential by -5--10 mV (conductivity range l-40 I&) (34). To avoid such fluctuations a base electrolyte (e.g. NaCI) could be added which leads to constant potentials in the conductivity range of 50-100 pS (34). These zeta potentials however at 50 pS are higher than in the emulsion and appear to be less useful in assessing the real situation in the emulsion bottle. Addition of a base electrolyte is meaningful in determining relative differences between emulsions because the measurements are independent of minor fluctuations in the conductivity of the distilled water. Zeta potential measurements in distilled water at 36 months (34) led again to values in the range obtained at 0 and 12 months. This supports the assumption that the increase is not due to chemical decomposition. In addition the chemical stability data (37) do not support a sufficient formation of lysolecithin to cause an increase of the potential by -10 mV. For example, a percentage of about 20% lysolecithin in the emulsifier film was found to lead to a zeta potential increase of -25 mV (38).

Conclusions The characterization data showed no batch to batch variation in the 10% emulsions, and little variation in the 20% emulsions. The latter was attributed to differences in the homogenization conditions due to an increased concentration of dispersed phase (oil). PCS data were very consistent when applying constant sample times of 25 ps instead of using the decay of the correlation function to l/e (15). Microscopy and LD data were in agreement. The microscopic analysis of undiluted fat emulsions provided comparable information and was more sensitive in detecting single large droplets. Microscopy is less costly and more easily available than a Laser Diffractometer or a Coulter Counter. From these considerations the use of microscopy can be recommended to hospital pharmacies to characterize emulsions and determine their stability. The Lipofundin MCT/LCT emulsions were physically stable over a period of 2 years. The size of the bulk population and the percentage of particles larger than 1 pm did not increase. The mean PCS diameter of the bulk population could be reproduced within a few nm over the 24 months of storage. Calculation of

AD proved suitable for monitoring minor formation of larger droplets in the 20% emulsions which could not be detected by microscopy and LD. This demonstrates the sensitivity and the potential of PCS. The lack of detection by microscopy indicates, however, that the size and number of larger droplets is low and of no clinical relevance. The reduction in microviscosity of the emulsifier film by displacement of LCT by MCT had no effect on the long-term stability.

Acknowledgement We would like to express our sincere thanks to Professor S. S. Davis for,the opportunity to conduct the two years stability study in his laboratories at the University of Nottingham.

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Submission date: 2 August 1991; Accepted after revision: 9 March 1993