Authors:
Marija Pavlovic 1 , Mirjana Antunovic 2 , Dusica
Mirkovic
2
, Aleksandar Petrovic
3
Institutions:
1
Department of Pharmacy, Faculty of Medicine,
University of Nis,
2
Institute of Pharmacy,
Military Medical Academy, Belgrade, 3 Institute of Histology and Embryology, Faculty of
Medicine, University of Nis, Republic of Serbia
Introduction: Total parenteral nutrition (TPN) purpose is to provide all nutrients according to individual nutritional needs of every patient whose digestive tract is non-functional or inaccessible
1,2,3
.
All-in-One system (all components are mixed in one bag), is TPN standard, as it maximizes nutrition support by tailoring nutritional formula for each patient requirements 2,3 .
The TPN admixtures are sterile, large volume, complex formulations, containing over 50 individual components
(e.g. amino acids, glucose, lipids, electrolytes, trace elements, vitamins and water)
3,4
. Due to the oil/water emulsion character of TPN admixture, and its multi-composition and chemically reactive nature, its stability can be easily disturbed by physicochemical factors
(e.g. low pH, high of concentration cations, trace elements) 3,5 . Lipid emulsions are thermodynamically unstable systems as two phases tend to separate over time
6
. Creaming can occur without a significant change in mean droplet size (MDS) diameter and TPN admixture can be administered if the fat is first dispersed by gentle shaking, but if coalescence or cracking are present it must not be administered to the patient
5
. Hospital pharmacist is responsible for physiochemical and microbiological stability and safety of
TPN admixture which is often extemporaneously prepared and given to patient right away
4
. However, during holidays and weekends, after being prepared, TPN admixtures need to be stored (at 2-8 °C), so their time-dependent stability and expiry date need to be known. Stability refers to TPN admixture being stable during its preparation, storage and administration
.
The most critical TPN admixtures stability parameter is lipid particle diameter, as lipid emulsion particles greater than 6
 m in diameter can potentially obstruct pulmonary arterioles and accumulate
5
.
Lipid droplets diameter must be less than 5
 m (Ph. Jug. IV request), and USP request are
MDS < 500 nm and that volume-weight percentage of fat globules (PFAT5) > 5
 m must not exceed 0.05%
7,8
. Different methods may be used to determine physical stability
(particle size and size distribution), but only in laboratory, not in daily clinical practice 5 .
Objectives: To manufacture TPN admixture
(with lipid emulsion which declaration claimed expiry date was in 6 months), determine its physical stability (lipid particles diameter and their distribution) in order to state its expiry date.
Materials and methods: The commercial preparations used for preparing the analyzed
TPN admixture were: Lipovenoes 10% and
Aminosol 15% (by Fresenius Kabii), Glucosi injectio 500 mg/ml, Glucosi infundibile 100 g/l, Natrii chloridi conc. ad infundibile 100 g/l, Kalii chloridi conc. ad infudibile 74, 5 g/l,
Calcii chloridi injectio 100 mg/ml, Magnesii sulfatis injectio 250 mg/ml (by Institute of pharmacy, Military Medical Academy). The
TPN admixture contained: amino acids 75 g, glucose 250 g, lipids 50 g, electrolytes (90 mmol Na
+
, 50 mmol K
+
, 4 mmol Mg
2+ and 4 mmol Ca
2+
); total volume was 2009,4 ml.
Following protocols for component mixing order and preparation in laminar air – flow environment, TPN admixture was aseptically prepared into ethyl-vinyl acetate (EVA) bag, and after slow homogenization, physical stability of TPN admixture was analyzed just

after preparation (0h) and after 24, 48, and
72h. The TPN admixture was kept at 2-8 °C between taking samples that were diluted with sterile water in ratio 1:3 (v/v), and 2
 l were put on microscopic slide, covered with cover slip and immersion oil, and analyzed on light microscope equipped with digital camera.
Sample of intact Lipovenoes 10% was previously taken and analyzed, too. For each period, several digital pictures were taken
(1000x magnification), and analyzed by
Image J, that was set to determine lipid droplets diameter; 100 droplets (including aggregated forms, if present) per picture were chosen by grid option for measuring. The data were sorted to calculate the distribution of lipid particles diameter.
Results and discussion: The maximal lipid droplets diameter detected in intact lipid emulsion (Lipovenoes 10%) was close to 3,5
μm, while the most frequent (69,72%) lipids droplets were from 0,5 to 1 μm (Table 1.). The highest percent (71,4%) of same lipid droplets diameter range is present in TPN admixture right after its preparation, too. Commercially available lipid emulsions have a MDS of about 0.25–0.5
 m in diameter, like natural chylomicrons
5
, but the emulsion we used had
MDS diameter of 0.93±0.50 μm and 0,97% of larger droplets (3-4 μm), most likely because of used emulsion age as there were 6 months left before its expiry date. Previous EVA bag homogenization of TPN admixture had caused
Table 1 .
Percentage of lipid particles diameter in time
0-0,5
0,5-1
1-2
2-3
3-4
4-5
> 5 x±SD
Lipid particle diameter
(μm)
Intact lipid emulsion:
Lipovenoes
10%
TPN admixture
Time (h)
0+24 0+48 0 0+72
3,91
69,72
21,82
3
71,4
23
2,8
71,38
21,37
2,41
62,67
30,37
1,91
59,87
31,86
3,58
0,97
0
0
2,4
0,2
0
0
3,38
0,48
0,59
0
1,79
1,79
0,97
0
2,55
0,95
0,95
1,91
0.93±0.50 0.93±0.39 0,94±0,49 1,06±0,56 1,18±0,93 percent of 3-4
 m droplets to be less at start.
Although MDS diameter in TPN admixture was similar during first 24h, the particles from 4 to 5
 m are detectable after 24h, and their percent rises over time, plus more and more lipid droplets diameter grows to 2
 m.
Despite of larger lipid particles growth, this
TPN admixture fulfils pharmacopoeias’ requests during first 48h regarding lipid particle diameter (<5 μm)
7,8
. However, distribution of lipid particles diameter after
72h, shows further decrease of small lipid droplets (<1
 m) percents, plus aggregated lipid forms that have exceeded 5
 m in diameter are detected (1,91%) so pharmacopoeias’ requests 7,8
are not fulfilled.
These results confirm this TPN admixture is physically unstable and unsafe for clinical use after 72h, and its expiry date would be 48h.
Conclusions: The lipid particle diameter distribution is an important parameter for determining physical stability of emulsion systems. The age of used emulsion should be considered if preparing a TPN admixture that would not be used the same day. We showed that in that case, the TPN admixture prepared by hospital pharmacist could be used during the following 48h, if kept at 2-8 C.
References :
1. ASPEN Board of Directors and the Clinical Guidelines Task
Force. Guidelines for the use of parenteral and enteral nutrition in adults and pediatric patients: administration of specialized nutrition support. JPEN 2002; 26: 18S.
2. Singer P., et al. ESPEN Guidelines on Parenteral Nutrition:
Intensive care. Clinical Nutrition 2009; 28: 387–400.
3. Sobotka L., et al. Basics in clinical nutrition, 3rd edition. Prague:
House Galén, 2004; 253-274.
4. Barnett M.I., et al. Basics in clinical nutrition: Parenteral nutrition admixtures, how to prepare parenteral nutrition (PN) admixture. e-
SPEN, the European e-Journal of Clinical Nutrition and Metabolism,
2009; 4 (3): e114-e116.
5. Pertkiewicz M., et al. Basics in clinical nutrition: Stability of parenteral nutrition admixtures. e-SPEN, the European e-Journal of
Clinical Nutrition and Metabolism 2009; 4: e117–e119.
6 . Sinko PJ. Martin’s Physical Pharmacy and Pharmaceutical
Sciences, fifth ed. Lippincott Williams & Wilkins, Philadelphia, PA,
2006; 509–519.
7 . Pharmacopoea Jugoslavica. Editio Quarta. Volumen II. Belgrade:
Savezni zavod za zaštitu i unapređenje zdravlja, 1984 .
8 . United States Pharmacopoeia 26th. Rockville: The United States
Pharmacopoeal Convention, 2003.