Journal of Parenteral and Enteral

Nutrition http://pen.sagepub.com/

The Stability of Amikacin, Gentamicin, and Tobramycin in Total Nutrient Admixtures

L. Bullock, J.H. Clark, J.F. Fitzgerald, M.R. Glick, B.G. Hancock, J.C. Baenziger and C.D. Black
JPEN J Parenter Enteral Nutr 1989 13: 505
DOI: 10.1177/0148607189013005505

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 The Stability of Amikacin, Gentamicin, and Tobramycin in Total
Nutrient Admixtures

L. BULLOCK,* PHARM.D., J. H. CLARK,† M.D., J. F. FITZGERALD,‡ M.D., M. R. GLICK,§ PHD.,

B. G. HANCOCK,¶ M.SC., J. C. BAENZIGER,∥ M.D., AND C. D. BLACK,a PH.D.
From the *Departments of Pediatric Gastroenterology, ‡Pediatrics, and ∥ Pathology, Indiana University School of Medicine, the †Department of
Pediatrics, Medical College of Georgia, the Departments of §Pathology and ¶Pharmacy, Wishard Memorial Hospital, Indianapolis, Indiana, and
the a
Department of Pharmacy, Purdue University, West Lafayette, Indiana

ABSTRACT. Amikacin (A), gentamicin (G), and tobramycin
(T) were added to eight different total nutrient admixtures
(TNA) with varying concentrations of dextrose, amino acid,
and fat emulsion to determine drug and emulsion stability. All
TNA were prepared aseptically and stored at room temperature
under normal room lighting for 12 hr before drug addition. One
volume of each drug was added to an equal volume of each of
the eight TNAs to simulate 1:1 piggyback contact volumes.
Samples were left at room temperature for 6 hr. Drug concen-
trations were analyzed by fluorescence polarization immunoas-
say. TNA/drug admixtures were pH tested and visually in-
spected before and after centrifugation in microhematocrit
tubes, noting signs of emulsion stability at 1 and 6 hr. Emulsion
particle size was determined at 1 and 6 hr using interference
contrast microscopy. All three drugs retained their immuno-
reactivity in all TNAs for at least 6 hr. G and T were stable in
all eight TNAs for at least 6 hr with no significant effect on
emulsion particle size or stability after centrifugation. A was
incompatible with all eight TNAs, resulting in visual breaking
of all emulsions within 1 hr. Therefore, G and T, but not A,
can be administered via piggyback method with the eight TNAs
tested if the infusion is completed within 6 hr. (Journal of
Parenteral and Enteral Nutrition 13:505-509, 1989)

Infants and malnourished children often require a total
nutrient admixture (TNA); a parenteral solution con-
taining a fat emulsion dextrose and amino acids. This
single solution has an obvious advantage over the tradi-
tional two container system in patients with limited
venous access. The TNA system also allows continuous
24 hr infusion of small quantities of fat emulsion which
has been shown to be better tolerated in some neonates
with decreased serum triglyceride levels with continuous
infusions.’ These TNA solutions appear to be cost effec-
tive in pediatric inpatient populations.’
Limited intravenous access in neonates and infants
MATERIALS AND METHODS
Eight TNA solutions with varying concentrations of
dextrose, amino acids, and intravenous fat emulsion were
prepared aseptically under a laminar air-flow hood (Ta-
ble I). All solutions contained identical concentrations
of electrolytes, minerals, vitamins, and trace elements
(Table II). Concentrations of additives were within
ranges previously found compatible’ (personal commu-
nication, KabiVitrum, Inc, Alameda, CA). Standard
pharmacy compounding procedures for TNA solutions
were employed. Admixtures were prepared in 250-ml

frequently necessitates the temporary discontinuation of
TNA while antibiotics and other medications are infused.
The increased line manipulation increases the potential
for sepsis, and results in decreased caloric intake and a
risk of hypoglycemia. The loss of total calories can be
substantial if several intravenous antibiotics are admin-
istered. It would be beneficial to the patient if these
medications could be administered via Y site injection
without interruption of the TNA. We have, therefore,
evacuated glass containers and stored at ambient tem-
perature (20 to 22°C) under normal room light for 12 hr
before antibiotics were added, in order to simulate the
actual use condition.
Amikacin sulfate, 250 mg/ml (Amikin, Bristol Labo-
ratories, Syracuse, NY), gentamicin sulfate, 40 mg/ml
(Elkins-Sinn, Inc, Cherry Hill, NJ), and tobramycin
sulfate, 40 mg/ml (Nebcin, Eli Lilly, Inc, Carolina, PR)
were acquired from commercially available sources for

examined the stability of three commonly used antibiot- use in this experiment. Twelve hr after admixture prep-

ics in TNA solutions. The objectives were as follows; (1) aration 10-ml aliquots of each drug were added to equal
To determine the stability of TNAs when drugs are added aliquots of each TNA in sterile flasks and shaken to mix
to simulate piggyback administration, and (2) to deter- (time 0). The ratio of drug to TNA was 1:1 to simulate4
=

mine the stability of amikacin, gentamicin, and tobra- piggyback administration of the drug into the TNA.4
mycin in selected TNA solutions. Aliquots of each TNA-drug admixture were withdrawn
at 1 and 6 hr and stored at -15°C for subsequent analysis
of drug concentration.
Separate aliquots of each TNA-drug admixture were
Reprint requests: Ms. Linda Bullock, Department of Pediatric Gas- analyzed for lipid particle size and distribution at 1 and
troenterology, Indiana University School of Medicine, 702 Barnhill 6 hr by interference contrast microscopy using a Zeiss
Drive, Indianapolis, IN 46223. Standard RA microscope (Carl Zeiss CO, Ltd, Leondro,
505

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506
TABLE I and areas of no turbidity, ie, clear areas. The percent of
Total nutrient admixtures tested
linear distance for each of the three areas in each sample
was measured by the visual microhematocrit technique.

Drug concentrations are analyzed in singlet by fluo-

rescence polarization immunoassay (Abbott TDX, Ab-
bott Diagnostics, North Chicago, IL). One- and 6-hr
samples were thawed to room temperature and mixed
thoroughly before analysis. All TNA-drug admixtures
and control drugs were diluted to a final dilution of 1:4980
TABLE II using an automated dilutor (Cavro Scientific Instru-
Solution additives ments, Sunnyvale, CA) and a tris-HCI buffer, pH 7.9,
0.055 M/liter (Syva Company, Palo Alto, CA). Before
assay procedures, the equipment was calibrated with
standards of known concentrations of each drug. The
amount of drug recovered from the samples as measured
by the immunoassay was compared to the anticipated
recovery of a 1:1 mixture of the drug with TNA. The
*
Sodium chloride, sodium lactate, or sodium acetate. percent recovery was calculated for each sample, and the
t Potassium chloride or potassium phosphate. mean and standard deviation was calculated for each
$ Calcium gluconate. group of samples.
§ Magnesium sulfate.
11 Zinc sulfate.
lfBiotrace 4R. RESULTS

Results of drop size and particle distribution at 1 and

CA) equipped with a polarizing filter, interference-con- 6 hr are presented in Tables III and IV, respectively.
trast condenser, and an eye-piece micrometer. One sam- Amikacin was found to break all eight emulsions within
ple was withdrawn from each study and control solution 1 hr with visual signs of oil particles floating on the
and slides were prepared in duplicate using a 1:20 dilution admixtures. Creaming was not observed in the other
of admixture in 0.5% Carbopol 940 to increase viscosity admixtures. Admixtures containing gentamicin and to-
and minimize Brownian motion. The samples were bramycin had less than 4% of the drops greater than 1
viewed at 1000 x magnification. The drops were counted Am at 6 hr. Control admixtures contained less than 2%
as the eye piece containing the linear scale was slowly
turned through full 360° turn. All drops that were
a
TABLE III
greater than or to V2 jim were counted during the
equal Drop size and particle distribution of TNA at 1 hr
first sweep. A second sweep of the same field was made
to count drops larger than 1 Am. This counting procedure
was repeated for 10 fields of each TNA-drug admixture
as well as control TNAs. The results are reported in

percent of drops greater than 1 jim which was calculated

using the formula:

The samples were visually inspected at 1 and 6 hr after AA, amino acid-FreAmine III 8.5%. D, Dextrose injection 70%, USP; IL,
Intralipid 20%; 1&dquo; higher concentration; y, lower concentration.
drug addition for signs of emulsion instability, and the t Solutions were so extensively disrupted that particle size analysis could not
be determined.
pH measured using a standard pH meter (Model 3500,
Beckman Instruments, Inc, Altex, San Ramon, CA).
Solutions without drug served as controls and were tested TABLE IV
similarly. Admixtures were considered stable by visual Drop size and particle distribution of TNA at 6 hr

inspection if they exhibited an opaque nonreflecting

surface. Unstable admixtures were characterized by the
presence of clear yellowish oil droplets on the emulsion
surface. Creaming was also monitored and admixtures
were shaken if creaming occurred.
One sample of each TNA-drug admixture and each
TNA without drug were withdrawn in microhematocrit
tubes at the 1- and 6-hr sampling times and exposed to
a 11,000 x g centrifugal field for 5 min using a standard
blood hematocrit centrifuge (IEC, Model MB, American *
Refer to Table III for explanation.
Dade, Irvine, CA). The microhematocrit tubes were then t Solutions were so extensively disrupted that particle size analysis
inspected for areas of dense turbidity, transitional area could not be determined.

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of the drops greater than 1 ,um while admixtures contain-
ing amikacin contained more than 11% of the particles
greater than lgm at both sample times. Some amikacin-
TNA samples were so extensively disrupted that particle
size could not be quantitated. Results of paired t-tests
indicated that drop size and particle distribution was not
significantly changed from t = 0 to t = 6 hr for amikacin,
gentamicin, or tobramycin with p values of 0.258, 0.541,
and 0.108, respectively.
The pH of each sample was generally unchanged or
increased slightly over the 6-hr period. Solutions con-
taining amikacin demonstrated a significant change in
pH compared to TNA without drugs (p value 0.003). =
Results of pH measurements are illustrated in Table V.
The mean pH of control TNAs at 1 and 6 hr was 6.19 ±
0.09 and 6.21 ± 0.09, respectively.
Drug assay results are reported in mean percent recov-
ery of drug (Table VI). The expected results would be
that 100% of the drug was recovered if the drug were
stable in the TNA. These data suggest that each drug
tested can be expected to maintain its immunoreactivity
after 6 hr of admixture with the TNA solutions tested.
Results of fluorescent polarization immunoassay for re-
covery of gentamicin and tobramycin were within re-
ported ranges for the instrumentation used when com-
pared to results of 1881 laboratories using fluorescence
polarization immunoassay techniques.’ Amikacin recov-
ery was greater than 111% for 17 of 18 samples at both
sampling times. This exceeds the variability of the in-
strumentation used and was felt to be due to &dquo;oiling out&dquo;
with oil particles rising to the surface of the emulsion
resulting in a higher concentration of drug in the aqueous
phase of the admixture. Results of paired t-tests indicate
that there was a significant increase in amikacin concen-
tration over time (p < 0.05).
Figure 1 is representative of the microhematocrit cen-
trifugation results. At both sample times, all amikacin-
TNA solutions had no transitional area, a 3% opaque
area, and the remaining 97% of the area clear.
DISCUSSION
This study indicates that gentamicin and tobramycin
can be piggybacked into the eight TNAs tested without
affecting the stability of the emulsion or the stability of
TABLE V
Mean pH determinations
TABLE VI
Results of drug assays
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507
FIG. 1. Sample results of microhematocrit centrifugation.
the drugs, if the infusion is completed within 6 hr.
Amikacin, on the other hand, caused immediate &dquo;oiling
out&dquo; of the emulsion and should not be piggybacked into
the TNA solutions tested.
The TNA solutions that were tested were selected to
represent the maximum and minimum concentrations of
dextrose, amino acids, and fat emulsions that are known
to be compatible, or are routinely used in our hospital.
The dextrose concentration studied was either 10%
which is the minimum concentration used for peripheral
nutrition, or 25%, which is commonly provided centrally.
The electrolytes, minerals, vitamins, and trace elements
were added in the maximum concentrations previously
found compatible with TNA solutions (personal com-
munication, KabiVitrum, Inc, Alameda, CA). Parenteral
nutrition solutions are prepared individually in our hos-
pital according to each patient’s specific needs. Testing
the maximum and minimum concentrations of macro-
nutrients should account for the total range of TNA
solutions prepared in our hospital. The electrolyte con-
centrations exceeded doses generally required in pediat-
ric parenteral nutrition.
The delivery of intravenous medications is affected by
many factors, including infusion rate, site of injection,
characteristics of the drug, and the type of infusion
system used. A 6-hr analysis time was chosen because
studies have shown that the time of actual drug delivery
to the patient is much longer than the time predicted.6-9
The discrepancy between actual and predicted delivery
time appears to be most obvious with low intravenous
flow rates, used commonly in neonates. Gould and
associatesl° found that the actual time required for 95%
delivery of a 10-mg dose of gentamicin via Y site injection
at a flow rate of 3 ml/hr was 3.33 hr, 40 times the
predicted delivery time of 5 min. They further reported
that in one observation 30% of the gentamicin dose was
found at the Y site 8 hr after injection with the intrave-
nous flow rate at 3 ml/hr. Studies by Nahata et al6
supported the findings of Gould et al. 10 They found that
only 85% of a dose of chloramphenicol succinate was
delivered after 6 hr when the intravenous flow rate was
508
5 ml/hr. These studies demonstrate the need to deter-
mine drug compatibility with TNA solutions over an
extended period of time. Six hr was selected for this
study to determine the consequences of TNA-drug ad-
mixture over a period of time beyond that predicted for
drug delivery. The authors are not suggesting that the
drugs tested be administered over 6 hr.
The predominant factors affecting emulsion stability
are pH and increased electrolyte concentration. 11,12
These factors will result in increased particle size and a
greater potential for emulsion breakdown. Amino acids
are thought to stabilize TNA solutions by buffering the
system against pH changes caused by other additives.&dquo;
Hardy et a1.14 reported visible stability of four TNA
solutions with high and low concentrations of amino
acids and dextrose; yet, we felt that these variables
should be included in our study. Neonatal TNA solutions
are frequently initiated with low concentrations of amino
acids which could decrease the protective buffering ca-
pacity of the system, and lead to a decrease in pH when
concentrated dextrose solutions are added. Zerinque et
al.15 reported a loss of emulsion stability when the pH
dropped below 5.0. Other investigators have reported
that a pH less than 5.5 may cause stability problems
with TNA solutions.&dquo; All eight TNA solutions tested
had a pH of 5.0 or less when mixed with amikacin. These
emulsions showed visible signs of emulsion breakdown
within 1 hr, and an increased percentage of drops greater
than 1 wm. The importance of the solution pH was
demonstrated in a subsequent study in which the pH of
undiluted amikacin was increased to 5.92 with sodium
hydroxide, then added in a one to one ratio (vol-
ume :volume) to a TNA. The amikacin/TNA admixture
was visibly stable at 1 and 6 hr. Particle size and drug
concentration were not determined. Compatibility of
amikacin sulfate 50 mg/ml (Amikin, Bristol Laborato-
ries, Syracuse, NY) was tested in a TNA in the same
manner and it was found to be visually stable at 1 and 6
hr. The differences in compatibility of these two products
may be due to differences in pH, drug concentration, or
the concentrations of other additives. The mean pH of
50 mg/ml of amikacin and 250 mg/ml of amikacin were
4.31 ± 0.01 and 4.65 ± 0.02, respectively. It would seem
unlikely that 50 mg/ml of amikacin (pH 4.31) would be
more stable in TNA than 250 mg/ml of amikacin (pH
4.65), if the pH were the only destabilizing factor. This
was reinforced by the finding that the mean pH of the
gentamicin used in the study was 3.88 ± 0.03 and it was
found to be stable with all eight TNA solutions tested.
Amikacin sulfate solutions also contain sodium bisulfite
and sodium citrate. The concentration of each of these
additives is five times greater in 250 mg/ml of amikacin
compared to 50 mg/ml of amikacin. These additives in a
higher concentration (250 mg/ml of Amikin) may have
acted to destabilize the emulsion and cause oiling out.
This theory is supported by unpublished data suggesting
that citrate destabilizes fat emulsions (personal com-
munication, KabiVitrum, Inc, Alameda, CA). Citrate is
also known to complex with cations such as calcium. 16
Neither gentamicin nor tobramycin solutions for injec-
tion contain citrate.
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Results of microhematocrit centrifugation support the
visual and microscopic methods for determining emul-
sion stability. A stable emulsion would be expected to
have an area of dense turbidity, a transitional zone, and
a clear zone. After centrifugation an unstable emulsion
would be expected to consist of mostly clear (aqueous)
solution and a small dense area with no transitional zone
due to the oil droplets floating on top of the liquid phase.
The amikacin-TNA samples were found to separate into
a clear and dense area with 97 to 99% of the linear
distance being clear. All control emulsions had less than
50% of the linear distance clear.
The significance of emulsion particle size has been
established by reports of adverse reactions associated
with the infusion of particles greater than 6 Am in
size.17,18 The mean particle size of commercial fat emul-
sions is 0.4 to 0.5 J.Lm.19 This is within the range of
naturally occurring chylomicrons (0.025-1.0 Am). If fat
particles aggregate, larger particles are formed which
migrate to the surface, resulting in a cream layer. Cream-
ing is reversible with agitation, but when left unchecked
the larger particles in the cream layer could coalesce.
Coalescence is irreversible and leads to &dquo;oiling out&dquo; of
the emulsion with a visible oil layer floating on the
solution.
The TNA solutions admixed with gentamicin and to-
bramycin maintained stable particle counts during the
study period with 92 to 96% of the particles less than 1
Am in size, which is within the range of naturally occur-
ring chylomicrons. In contrast, the amikacin-TNA ad-
mixtures produced unstable emulsions with 11 to 15% of
the particles greater than 1 Am, maximum particle size
was not quantitated.
Vitamin and amino acid stability were not evaluated
in this study. Further studies are needed to determine
the availability of vitamins and individual amino acid
from total nutrient admixtures when gentamicin and
tobramycin are piggybacked into the solutions.
We conclude that gentamicin and tobramycin in the
concentrations studied are stable for piggyback admin-
istration into the eight TNA solutions tested, if the
infusion is complete within 6 hr. Amikacin, 250 mg/ml,
is incompatible with all eight TNA solutions tested and
should not be piggybacked into these solutions.
ACKNOWLEDGMENTS
This study funded in part by KabiVitrum, Inc,
was
want to thank Ms. Vicki L.
Alameda, CA. The authors
Haviland for her invaluable assistance in the preparation
of this manuscript.
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