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Wei et al
Table 2. Calibration Curves of Vancomycin and Gentamicin at 280 nm and 255 nm.
Component

Concentration range,
µg/mL

Calibration curve at
280 nm

Calibration curve at
255 nm

Vancomycin
Gentamicin

15.625-500
12.5-400

y = 0.0039x + 0.0107
y = 0.0011x + 0.0021

1
1

y = 0.0024x + 0.0133
y = 0.0047x + 0.0023

0.9999
1

because the relative standard deviations of the drug concentrations did not exceed 0.27%. A different set of calibration standards ranging from 15.625 to 250 µg/mL were prepared for
analyzing vancomycin from admixtures containing ethanol at
280 nm. A straight line (y = 0.0042x - 0.0031) was obtained
with a correlation coefficient (r2) value of 0.9999 (Table 2).
Chemical stability of drugs in admixtures was assessed by
measuring the drug content at 0, 24, 48, and 72 hours.

Antimicrobial Susceptibility Testing
Stability of antimicrobial activity was determined by KirbyBauer disk diffusion method according to the guidelines
established by the Clinical and Laboratory Standards Institute
(CLSI).11 To determine antibacterial activities of gentamicin
and vancomycin, reference strains of Escherichia coli ATCC
25922 and Staphylococcus aureus ATCC 25923 were used,
respectively. Because a number of samples contained both
vancomycin and gentamicin, a clinical strain of S aureus
resistant to gentamicin but susceptible to vancomycin was
obtained from the University of Toledo Medical Center and
was evaluated in all samples containing vancomycin with
and without gentamicin.
The Mueller-Hinton agar (MHA) was prepared according
to the manufacturer's instructions. After autoclaving, the
agar was poured into flat bottomed petri dishes to give uniform depths of about 4 mm. The agar was allowed to cool to
room temperature and, unless used on the same day, stored in
the refrigerator (2°C-8°C). Bacterial colonies from overnight
growth were added to normal saline and adjusted turbidity to
equal to a 0.5 McFarland standard (1.5 × 108 colony-forming
units/mL). The surface of MHA plates was inoculated using
a sterile swab that was dipped into the adjusted bacterial suspension and streaked over the surface of the agar plates.
Antimicrobial susceptibility test disks containing gentamicin 10 µg and vancomycin 30 µg (BBL Sensi-Discs;
Becton Dickinson Biosciences, Franklin Lakes, NJ, USA)
were used. In addition, blank disks were impregnated with
required amounts of sample equivalent to the potency of the
standard gentamicin and vancomycin disks calculated based
on initial concentrations of vancomycin and gentamicin. The
standard and the sample disks were applied on the surface of
agar plates manually using sterile forceps in such a way that
each disk was at least 24 mm away from other disks and from
the edge of the petri plate to avoid contamination. The plates
were incubated in air at 35°C to 37°C for 24 hours before
measuring the zones of inhibition. All the tests were conducted in triplicate.

Antimicrobial activity was calculated by comparing the
zones of inhibition at 72 hours to the baseline values and
presented as a percentage of the baseline zones of inhibition.
These values were then compared using one-way analysis of
variance (ANOVA), and when statistically significant results
were obtained, Tukey post hoc test was conducted. A P value
≤0.05 was considered statistically significant. All statistical
tests were conducted using IBM SPSS Statistics 21 Software
(IBM Corp, Armonk, New York).

Results
Physical and Chemical Stability of Lock Solutions
At baseline, 24 hours, and 48 hours, all solution combinations
containing vancomycin (5 or 10 mg/mL) with either gentamicin (1 mg/mL) or ethanol (40%) in 4% sodium citrate under 4
different conditions remained visually clear, exhibiting no
precipitate or turbidity (all solutions were graded 0 at each
time point). Over 72 hours, all solutions except one combination maintained clarity measured by spectrophotometric
absorbance. At 48 hours, the admixture containing vancomycin (5 mg/mL) and ethanol (40% v/v) in a 4% sodium citrate
solution stored under 4 different conditions did not display
any turbidity but at 72 hours; this admixture showed an absorbance value >0.010 AU which indicated turbidity.
The pH of each admixture under 4 conditions maintained
pH within the acceptable margins during the 72-hour study
period. Chemical stability of gentamicin and vancomycin was
assessed and the mean concentrations ranged between 95%
and 105% of initial concentrations after 72 hours (Table 3).

Antimicrobial Susceptibility Testing
The average antimicrobial activity based on zones of inhibition is reported in Table 4. The mean antimicrobial activity of
vancomycin against S aureus ranged from 95% to 106% of
baseline zones of inhibition after 72 hours. The average
activity of gentamicin against E coli was 99% to 104% of the
baseline after 72 hours. Overall, no significant change was
observed for all 6 combinations of lock solutions under the 4
different storage conditions (P>0.05).

Discussion
Catheter lock solutions containing an antimicrobial agent
and anticoagulant solution have been used to reduce
CRBSIs and maintain patency. Although the most widely

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