Supplemental Methods
Procedures for drug extraction and quantification were developed based on previously reported
sample processing and HPLC methods. (1-4)
Chemicals used. Acetonitrile and methanol were HPLC-grade and purchased from VWR
(Radnor, PA). Ultrapure deionized water was generated using an Aqua Solutions Type I water
purification system (resistivity ≥18.2MΩ) and used in all applications. Piperacillin powder was
purchased from U.S. Pharmacopeia (Rockville, MD). Tazobactam sodium and oxacillin sodium
were purchased from Sigma-Aldrich (St. Louis, MO).
Sample Preparation-Free Drug. Free (protein-unbound) piperacillin and tazobactam were
determined using centrifugal ultrafiltration (UF).(4,5) 300uL of plasma sample was centrifuged
in Centrifree UF filters (30kD MWCO, Millipore) at 1500 x g for 30 min in a fixed-rotor
centrifuge.
Sample Preparation-Total Drug. The same sample was used for analysis of piperacillin and
tazobactam. 20 µg of internal standard (IS, oxacillin) was added to 200 µL plasma, which was
then drawn by vacuum through C18 solid-phase extraction (SPE) cartridges (Waters) previously
conditioned with methanol (MeOH), then hydrated with 0.067M phosphate buffer, pH 7.4.
Cartridges were washed with phosphate buffer (1 mL x 3) followed by 1 mL 5% MeOH in
phosphate buffer. Analytes were eluted with 100 µL, then 150 µL acetonitrile (MeCN),
evaporated under a stream of air, then reconstituted in 200 µL of the aqueous component of the
mobile phase (0.1M phosphate buffer, pH 3.0).
High-Performance Liquid Chromatography (HPLC). HPLC was performed with a Dionex 3000
HPLC system (Dionex, Thermo-Fisher Inc., Sunnyvale CA) with UV detection (wavelength =
210nm). A C18 stationary phase (Kinetix 75 x 4.6 mm, 2.6 µm/100A particles, Phenomenex)
with mobile phase flow rate of 1 mL/min was used. Piperacillin and tazobactam were both
determined on this system using two separate methods. Piperacillin. The chromatogram was
developed isocratically using a mobile phase of 0.1M phosphate buffer, pH 3.0 and acetonitrile
(75:25). Injection volume was 10µL and elution times were approximately 3.3 minutes
(piperacillin) and 8.7 minutes (IS). Tazobactam. The mobile phase consisted of a gradient of
0.1% phosphate buffer, pH 3.0 and acetonitrile. Initial conditions consisted of 5% acetonitrile,
which after 2 minutes was gradually increased to 50% over 10 minutes. Injection volume was
25µL and elution time was 2.3 minutes (tazobactam) and 10.2 minutes (IS).
Validation of Methods. For each analyte, separate calibration curves were constructed for
determination of free and total drug. Free drug measurements were calibrated against dilutions of
drug stocks in mobile phase. For both drugs, five to seven calibration standards were prepared in
parallel to samples by SPE (1-500µg/mL for piperacillin, 0.05-200µg/mL for tazobactam) or in
buffer (1.25-500 µg/mL for piperacillin, 0.1-40µg/mL for tazobactam). These were used to
construct calibration curves for total and free drug, respectively. Standards confirmed a linear
response in these ranges between analyte:internal standard peak area ratio and analyte
concentration (for total drug) or analyte peak area and analyte concentration (for free drug). A
validation standard (50 µg/mL piperacillin, 10µg/mL tazobactam) was used to monitor accuracy
between calibration runs. Within-/between-day variability was 1.1%/5.1% for 50 µg/mL
piperacillin, and 3.8%/15.7% for 10 µg/mL tazobactam.
1.
Denooz R, Charlier C. Simultaneous determination of five β-lactam antibiotics (cefepim,
ceftazidim, cefuroxim, meropenem and piperacillin) in human plasma by high-performance
liquid chromatography with ultraviolet detection. Journal of Chromatography B 2008;864(1–
2):161-67.
2.
McWhinney BC, Wallis SC, Hillister T, Roberts JA, Lipman J, Ungerer JPJ. Analysis of
12 beta-lactam antibiotics in human plasma by HPLC with ultraviolet detection. Journal of
Chromatography B 2010;878(22):2039-43.
3.
Di Giovamberardino G, Ferrannini M, Testore GP, Federici G, Pastore A. High
performance liquid chromatographic determination of plasma free and total tazobactam and
piperacillin. Journal of Chromatography B 2009;877(1–2):86-88.
4.
Bauer SR, Salem C, Connor MJ, Groszek J, Taylor ME, Wei P, Tolwani, AJ, Fissell,
WH. Pharmacokinetics and Pharmacodynamics of Piperacillin-Tazobactam in 42 Patients
Treated with Concomitant CRRT. Clinical Journal of the American Society of Nephrology
2012;7(3):452-57.
5.
Millipore. User Guide: Centrifree® Ultrafiltration Devices, For Research and In Vitro
Diagnostic Use [ cited 2013 October 31]; Available from:
http://www.millipore.com/userguides/files/centrifugal/$file/PR03523TR_RevA_English.pdf
Supplemental Table 1. Summary characteristics of the study subjects according to ARC score group (mean ± SD or median/IQR).
ARC score group
Low (0-6)
High (7-11)
5
8
40.0%
75.0%
0.29
Age (years)
58.0 ± 21.0
36.4 ± 11.8
0.08
Piperacillin dose (mg/kg)
42.1 ± 10.5
47.3 ± 19.1
0.55
Tazobactam dose (mg/kg)
5.1 (4.3, 6.4)
5.1 (3.9, 7.2)
0.81
N receiving 4g/500mg
1
4
N receiving 3g/375mg
4
4
98.0 (93.1, 193.8)
132.3 (98.8, 187.6)
0.42
SOFA score
5.4 ± 3.0
5.6 ± 2.1
0.77
APACHE II score
15.0 ± 5.2
15.5 ± 5.6
0.94
Number of Subjects
% Male
eGFR (mL/min/1.73m2)
P
Supplemental Table 2. Differences in piperacillin fT>MIC (%) were significant across a range of MIC values (N=13).
ARC ≤ 6
ARC ≥ 7
P
Piperacillin MIC (µg/mL)
8
16
1
2
4
100.0 ± 0.0
97.9 ± 6.5
0.34
100.0 ± 0.0
94.2 ± 13.2
0.26
100.0 ± 0.0
79.2 ± 19.9
0.02
99.9 ± 0.09
61.9 ± 21.4
0.002
92.9 ± 7.1
37.0 ± 15.1
< 0.0001
32
64
70.3 ± 21.2
11.8 ± 12.2
0.002
22.6 ± 29.6
1.3 ± 3.5
0.18
Supplemental Table 3. Potential dosing regimens, predicted efficacy, and drug costs for intermittently-delivered alternative
piperacillin dosing strategies for patients with high ARC scores (except where indicated).
Interval
Q4h
Q6h
Dose
Probability of fT>MIC
Daily Total
Estimated Daily
>50% (MIC 16 µg/mL)
Piperacillin
Drug Cost
2g
26.7%
12 g
$20.04
3g
47.6%
18 g
$30.06
4g
61.8%
24 g
$40.08
6g
79.9%
36 g
$60.12
3 g (low ARC score)
99.5%
12 g
$20.04
4 g (low ARC score)
100.0%
16 g
$26.72
3g
19.8%
12 g
$20.04
3 g (4h infusion)
68.6%
12 g
$20.04
4g
32.1%
16 g
$26.72
4 g (4h infusion)
86.2%
16 g
$26.72
6g
51.8%
24 g
$40.08
8g
64.1%
32 g
$53.44
Q8h
3 g (4h infusion)
59.0%
12 g
$20.04
4 g (4h infusion)
73.0%
16 g
$26.72
6g
26.1%
18 g
$30.06
8g
37.2%
24 g
$40.08
500 mg/h
46.0%
12 g
$20.04
583 mg/h
53.7%
14 g
$23.38
667 mg/h
59.6%
16 g
$26.72
750 mg/h
64.9%
18 g
$30.06
1000 mg/h
77.9%
24 g
$40.08
Continuous