Provision of Standardized All-in-One Parenteral Nutrition (AIO-PN) for Very Preterm Neonates: Evaluation at Room and Cold Temperatures.

INTRODUCTION: All-in-one parenteral nutrition (AIO-PN) is essential for patients with limited venous access, e.g. premature infants. So far, there are still some conflicting data related to the stability of AIO-PN. The aim of this study is to examine the physical stability and sterility of AIO-PN as it was being stored at room and cold temperature.
MATERIALS AND METHODS: AIO-PN contains dextrose, amino acid, lipid, sodium chloride, magnesium sulfate, potassium chloride, calcium gluconate, and vitamin. Formulation of AIO-PN was prepared based on the guidance of nutrition for 3-day-old preterm baby weighing 1000 g. The formulation of AIO-PN then was stored at room temperature (25.43ºC ± 0.54) and cold temperature (6.2ºC ± 2.04). RESULTS AND DISCUSSION: Based on the experiments, all formulas confidently fulfilled the sterile criteria, in which there is no microbial growth in the formula within 7 days. During those days, the droplet size of all formulas was under the range (<500 nm) with a good range of pH. However, during the process of storing under the room temperature, AIO-PN showed the reversible creaming starting on day 2 and the discoloration starting on day 4. Meanwhile, we found that there are no such physical changes in the formula within 7 days under the cold temperature.
CONCLUSION: This research confirmed that AIO-PN that is being stored at room temperature cannot be used starting on day 4, but the formulation storage under the cold temperature is still accepted within 7 days. Copyright:

INTRODUCTION

Currently, the demand of total parenteral nutrition (TPN) compounding in hospital increases.[1] Preterm infants are born during the growth of their body and brain; therefore, preterm birth may lead to high stress or catabolism.[2] In addition, the gastrointestinal system is not ready yet to suck, swallow, and obtain the nutrition required.[3] This brings the importance of parenteral nutrition support until the infants are able to suffice the nutrition from the breast milk or enteral nutrition.[4] Therefore, premature infants, especially with very low birth weight should receive parenteral nutrition (PN) to obtain optimum growth rate.[3] Provision of TPN containing glucose, amino acid, electrolyte as well as lipid is paramount significance for preterm babies to fulfill the nutrition including essential fatty acids and fat-soluble vitamins. However, those compositions of TPN are manufactured as single or double components. Each administration of parenteral nutrition through a different single line will be a burden since venous access is limited. Furthermore, a very young baby has to get restricted quantity. Therefore, establishment of a high concentrated solution in a low volume formulation is often preferred. In order to obtain those total components of parenteral nutrition in the minimum volume, hospital pharmacy must mix all the nutrition as all-in-one parenteral nutrition (AIO-PN).

An issue may arise with intravenous admixtures or AIO-PN associated with incompatibility or instability. Physical instability is a significant matter that often causes vascular microemboli and induces deathly effect. Lipid added in the parenteral nutrition along with other components may form a two-phase medication as creaming, coalescence, or cracking that cause larger particle size. Additionally, the interaction of molecule among the cation–anion and acid–base of each ingredient may induce instability. High temperature may increase the reaction faster. In fact, lipid added in one chamber and stored at room temperature is common in a local hospital. However, relevant studies reporting this situation are limited.

MATERIALS AND METHODS

The nutrition was obtained from hospital stock; those are 40% Dextrose (Otsuka), 5% Dextrose (Otsuka), Aminosteril® Infant 6% (Fresenius Kabi Combiphar), NaCl 3% (Otsuka), Lipofundin® 20% (Braun), Potassium Chloride injection 7,46% (Otsuka), Calcium Gluconate Injection (Ethica Pharmacy Industry), Magnesium Sulphate injection 20% (Otsuka), Nutrient Pad Set Standard TTC Media, Membrane Filter (Sartorius), and Peptone Water (OXOID).

Study design

This study has been done to fulfill the quality control of production of AIO-PN standard in hospitals. This research was conducted in vitro including visual detection, pH, particle size, and sterility. The formula has been provided by mimicking the production of parenteral nutrition in hospital under ambient temperature in a sterile room and Laminar Air Flow. To reduce any contamination during sampling, a 10-mL sample was taken once for pH, sterility, and also particle measurement every day. In addition, the sterility test has been done each day to confirm the sterility shown with no microbial growth on microbial culture.

Formula

This study investigated the standardized parenteral nutrition for premature infant weighing 1000mg. The composition of nutrients was based on a guidance of first, second, and third day of a very low weight premature baby birth as seen in Table 1.[5] The study of stability was carried out on third formulas (F1, F2, and F3).

Table 1

Formula of TPN for premature infants (weight 1000 g)[5]

Nutrients	Volume
	F1 (cc/day)	F2 (cc/day)	F3 (cc/day)
Amino acid 6%	25	33,33	33,33
Lipid 20%	5	7,5	7,5
NaCl 3%	—	6	6
KCl 7,4%	—	2	2
Ca gluconate 10%	10	10	10
MgSO4 20	0,36	0,36	0,36
Dextrose 5%	28,846	30,182	53,04
Dextrose 40%	10,794	10,63	7,77

F1 = the first day standard formulation in one chamber to fulfill water body volume (80 mL), F2 = the second day standard formulation in one chamber to fulfill water body volume (100 mL) and maximum electrolyte dose, F3 = the third day standard formulation in one chamber to fulfill water body volume (120 mL) and maximum electrolyte dose

Experimental design

Three ready formulas (F1, F2, and F3) were prepared in triplicate, then were stored in two conditions: room temperature (25.43oC ± 0.535) and cold temperature (6.2oC ± 2.038), using climatic chamber. Therefore, there were six formulas in the triplicate that were evaluated. The formulas were stored for 7 days and were controlled every day, by which the assays are as follows:

Visual detection

The clarity of formulas in the spike container was detected under light and dark background. The changes in color, turbidity, gas formation, or emulsion changes (creaming, cracking, and coalescence) were justified as instability.

pH testing

The pH of 5 mL aliquot samples of each formula was detected by using a calibrated-pH meter with specification ISFET pH electrode (Horiba, Germany) each day for 7 days. The pH changes >0.5 from the pH baseline were indicated as instability.

Particle size analyses

Particles were measured using LA-960 (Horiba, Germany) with Mie and Fraunhofer principles. Two different wavelengths were used inside the instrument: Solid state laser diode 650 nm (Red) and Solid State LED 405 nm (Blue). Smaller particles would be covered by shorter wavelength and larger particles would be covered by longer wavelength of laser. Solid state laser would be better than Helium Neon Laser (gas) in terms of brighter/intensity and energy efficiency. We used an external validation with NIST Traceable Standard Particles (PSL) 0.1, 1, and 10 μm. The 1 mL aliquot samples were put on the flow cell with ultrasonic probe attachment in order to homogenize and prevent agglomeration. Optimal speed was used to adjust the particle circulation leading to conclusion that larger particle than 500 nm was considered as instability.

Sterility

The sterility was measured using a validated 0.45μm membrane filtration method and then transferred to nutrient pad set standard TTC media. No microbial growth specifies as sterile condition

RESULTS

Table 2 shows that the formula of AIO-PN was physically stable and sterile for 4 days and 7 days under room and cold temperatures, respectively. AIO-PN under both temperatures showed creaming process, but it was reversible after shaking. Instability of AIO-PN under room temperature was performed with discoloration since day 4. Meanwhile, AIO-PN under cold temperature was able to maintain the physical stability until day 7. There is no significant change in the particle and pH that showed instability. Furthermore, there is no microbial growth observed on the three formulas in either cold or room temperature.

Table 2

Result of control quality of AIO-PN in the specific storage temperature

Testing period	Temperature	Visual detection			Particle size			pH			Microbial growth
		F1	F2	F3	F1	F2	F3	F1	F2	F3	F1	F2	F3
Day 1	Room	White, milky	White, milky	White, milky	344.97 ± 3.700	314.60 ± 2.307	341.70 ± 3.915	5.86 ± 0.012	6.06 ± 0.087	5.90 ± 0.026	No	No	No
	Cold	White, milky	White, milky	White, milky	351.30 ± 7.227	313.23 ± 1.159	338.57 ± 2.228	6.06 ± 0.087	5.75 ± 0.038	5.84 ± 0.012	No	No	No
Day 2	Room	Slight and reversible creaming	Slight and reversible creaming	Slight and reversible creaming	317.73 ± 0.777	322.63 ± 2.593	356.20 ± 4.244	5.91 ± 0.015	5.76 ± 0.015	6.02 ± 0.036	No	No	No
	Cold	White, milky	White, milky	White, milky	344.90 ± 0.800	356.63 ± 2.122	475.57 ± 2.442	5.63 ± 0.049	6.04 ± 0.030	6.03 ± 0.036	No	No	No
Day 3	Room	Slight and reversible creaming	Slight and reversible creaming	Slight and reversible creaming	292.37 ± 1.656	334.40 ± 1.353	351.27 ± 6.704	6.03 ± 0.036	5.94 ± 0.068	6.16 ± 0.0416	No	No	No
	Cold	Slight and reversible creaming	Slight and reversible creaming	Slight and reversible creaming	329.90 ± 3.951	300.03 ± 6.058	307.37 ± 1.955	5.52 ± 0.098	6.04 ± 0.030	6.03 ± 0.036	No	No	No
Day 4	Room	Yellowish, oily in the bottle	Yellowish, oily in the bottle	Yellowish, oily in the bottle	585.23 ± 2.970	334.37 ± 2.984	453.23 ± 2.579	5.71 ± 0.053	5.66 ± 0.065	6.12 ± 0.015	No	No	No
	Cold	Slight and reversible creaming	Slight and reversible creaming	Slight and reversible creaming	349.00 ± 0.436	342.43 ± 0.929	319.67 ± 0.643	6.12 ± 0.015	6.04 ± 0.026	6.10 ± 0.031	No	No	No
Day 5	Room	Yellowish, oily in the bottle	Yellowish, oily in the bottle	Yellowish, oily in the bottle	587.53 ± 12.266	352.27 ± 8.693	766.20 ± 2.227	5.46 ± 0.091	5.68 ± 0.047	5.76 ± 0.040	No	No	No
	Cold	Slight and reversible creaming	Slight and reversible creaming	Slight and reversible creaming	335.87 ± 2.829	361.13 ± 1.007	324.87 ± 4.010	6.13 ± 0.035	6.15 ± 0.101	6.16 ± 0.040	No	No	No
Day 6	Room	Yellowish, oily in the bottle	Yellowish, oily in the bottle	Yellowish, oily in the bottle	317.83 ± 1.069	318.77 ± 0.635	305.93 ± 2.485	5.80 ± 0.015	5.70 ± 0.068	5.98 ± 0.064	No	No	No
	Cold	Slight and reversible creaming	Slight and reversible creaming	Slight and reversible creaming	352.10 ± 3.727	362.20 ± 0.283	323.03 ± 1.193	5.90 ± 0.015	5.98 ± 0.064	6.08 ± 0.040	No	No	No
Day 7	Room	Yellowish, oily in the bottle	Yellowish, oily in the bottle	Yellowish, oily in the bottle	332.67 ± 2.055	766.57 ± 12.169	291.37 ± 3.179	5.4 ± 0.075	5.85 ± 0.040	5.78 ± 0.090	No	No	No
	Cold	Slight and reversible creaming	Slight and reversible creaming	Slight and reversible creaming	369.20 ± 1.136	303.63 ± 0.603	325.70 ± 1.153	5.82 ± 0.006	6.22 ± 0.062	6.15 ± 0.062	No	No	No

F1 = standard formula for preterm baby weighing 1000 g in the first day, F2 = standard formula for preterm baby weighing 1000 g in the second day, F3 = standard formula for preterm baby weighing 1000 g in the third day

DISCUSSION

This study found that the three formulas of AIO-PN are stable under cold temperature up to 7 days. Even by using different formulas, this study confirmed the findings of Bouchoud that AIO-PN is stable until more than 7 days under cold temperature.[6] Moreover, parenteral nutrition for preterm babies has a higher concentration than other parenteral nutrition; however, this AIO-PN maintains its physical stability. Even though the creaming process was seen, it is commonly not a problem; it will be reversible by shaking and maintaining the particle size consistency. Parenteral emulsion will be unstable when there are coalescence and cracking, following the particle globule that grows larger.[7] Meanwhile, in this study the smaller particle size is in a good range (<500 nm). Emulsion droplet size less than 500 nm will not be dangerous as it is smaller than the smallest vasculature (400 nm). This is possibly because the particles circulate freely in a tiny capillary; therefore, it will not trap and induce emboli in a blood vessel. A recent study stated that high concentration of amino acid in this formula might increase buffer effect; therefore, the formula will be more stable.[8] This is also confirmed with the value of pH in a range 5–6. Degradation of AIO-PN is commonly realized from the decrease in pH. Lipid added to AIO-PN may degrade by forming of fatty acid that decrease the pH into more acid.[8] Therefore, the pH value of PN in a range 5–6 indicates the stability; in contrast, the pH that is less than 5 specifies free fatty acid formation being followed with larger globule.[89]

This study also identified that the AIO-PN under room temperature was less stable and able to maintain its stability only for 3 days storage. Emulsion might be adhesive and adsorbed into the container during storage.[10] Instability was justified from the visual changes as yellowish colour start from day 4. To the best of our knowledge, no study was done under room temperature. However, this study is relevant for hospitals that have no enough storage for TPN. Moreover, the administration of TPN in the ward is also under room temperature, so this finding will show how long the TPN will be stable during administration.[111213] This finding is different from the guideline that stated AIO-PN should not be used after 24h.[12] The temperature theoretically increases chemical reaction and hydrolysis that will cause instability. In terms of this situation, the different humidity may influence the stability.

According to the sterility test, this study acknowledged that AIO-PN mixture was sterile according to the sterility test of aliquot sample taken in each day up to 7 days. However, in practice, the sterility of intravenous admixture depended on the quality of management system in production unit. However, aseptic preparation may still preserve the contamination. Meanwhile, in practice, it is troublesome to test sterility in every single bag. Therefore, it is critical to check the visible sign of contamination in every single aseptic product prior to use.

This shows that the production of parenteral nutrition under Laminar Air Flow and HEPA filter in a local hospital is acceptable.[15] As a previous study, mixing the AIO-PN in a hospital maintained the stability either in two or one chamber formulation.[15] An issue may arise regarding the safe administration of AIO-PN. The provision of AIO-PN may reduce the necessity of intravenous line. Moreover, the result of the osmometer showed that osmolarity of this AIO-PN is lower than 850 mOsm (F1: 700–800 mOsm, F2: 600–700 mOsm, and F3: 500–600mOsm). According to the European Society for Clinical Nutrition and Metabolism (ESPEN), injection formula which has osmolarity less than 850 mOSm may be delivered through peripheral routes, although, the central routes is another choice for parenteral nutrition and being longer used.[14] This finding had validated what hospital had done related to the AIO-PN production, in which it will possibly administer within days through peripheral or central route access.

CONCLUSION

This study concluded that one chamber of AIO-PN maintained the original formulation for 3 days under room temperature and 7 days under cold temperature. This was confirmed visually and by the particle size and pH.

Limitations and future directions

Quality control of this AIO-PN was performed on physical matter; chemical assay was limited on pH. In the future, there is a need to assay chemical changes including the concentration and degradation.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.