Development and validation of a UPLC-MS:MS method to monitor cephapirin excretion in dairy cows following intramammary infusion

Development and Validation of a UPLC-MS/MS Method
to Monitor Cephapirin Excretion in Dairy Cows following
Intramammary Infusion
Partha Ray1
*, Katharine F. Knowlton1
, Chao Shang2
, Kang Xia2
1 Department of Dairy Science, Virginia Polytechnic Institute and State University, Blacksburg, Virginia, United States of America, 2 Department of Crop and Soil
Environmental Sciences, Virginia Polytechnic Institute and State University, Blacksburg, Virginia, United States of America
Abstract
Cephapirin, a cephalosporin antibiotic, is used by the majority of dairy farms in the US. Fecal and urinary excretion of
cephapirin could introduce this compound into the environment when manure is land applied as fertilizer, and may cause
development of bacterial resistance to antibiotics critical for human health. The environmental loading of cephapirin by the
livestock industry remains un-assessed, largely due to a lack of appropriate analytical methods. Therefore, this study aimed
to develop and validate a cephapirin quantification method to capture the temporal pattern of cephapirin excretion in dairy
cows following intramammary infusion. The method includes an extraction with phosphate buffer and methanol, solid-
phase extraction (SPE) clean-up, and quantification using ultra performance liquid chromatography-tandem mass
spectrometry (UPLC-MS/MS). The LOQ values of the developed method were 4.02 mg kg21
and 0.96 mg L21
for feces and
urine, respectively. This robust method recovered .60% and .80% cephapirin from spiked blank fecal and urine samples,
respectively, with acceptable intra- and inter-day variation (,10%). Using this method, we detected trace amounts (mg
kg21
) of cephapirin in dairy cow feces, and cephapirin in urine was detected at very high concentrations (133 to 480 mg
L21
). Cephapirin was primarily excreted via urine and its urinary excretion was influenced by day (P = 0.03). Peak excretion
(2.69 mg) was on day 1 following intramammary infusion and decreased sharply thereafter (0.19, 0.19, 0.08, and 0.17 mg on
day 2, 3, 4, and 5, respectively) reflecting a quadratic pattern of excretion (Quadratic: P = 0.03). The described method for
quantification of cephapirin in bovine feces and urine is sensitive, accurate, and robust and allowed to monitor the pattern
of cephapirin excretion in dairy cows. This data will help develop manure segregation and treatment methods to minimize
the risk of antibiotic loading to the environment from dairy farms.
Citation: Ray P, Knowlton KF, Shang C, Xia K (2014) Development and Validation of a UPLC-MS/MS Method to Monitor Cephapirin Excretion in Dairy Cows
following Intramammary Infusion. PLoS ONE 9(11): e112343. doi:10.1371/journal.pone.0112343
Editor: Bernhard Kaltenboeck, Auburn University, United States of America
Received July 1, 2014; Accepted October 10, 2014; Published November 6, 2014
Copyright: ß 2014 Ray et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted
use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: The authors confirm that all data underlying the findings are fully available without restriction. All relevant data are within the paper and its
Supporting Information files.
Funding: This activity was funded, in part, with an integrated, internal competitive grant from Virginia Agricultural Experiment Station, Virginia Cooperative
Extension, and the College of Agriculture and Life Sciences at Virginia Tech. The funders had no role in study design, data collection and analysis, decision to
publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* Email: drray@vt.edu
Introduction
Antibiotics are administered to livestock therapeutically to treat
bacterial infections and subtherapeutically for disease prevention
and growth promotion. Administered antibiotics are distributed in
tissues (liver, kidney, and muscle), secreted in milk [1–3], and
eventually excreted in feces and urine [2]. Antibiotic residues in
human consumable food products can pose a threat to human
health by causing toxicity or facilitating antibiotic resistance [4,5],
so maximum tolerances of antibiotics in food products of animal
origin are established and monitored. Antibiotic residues in milk or
meat are monitored by simple screening tests or kits designed
based on microbial- or immuno-assays.
While edible products from animals treated with antibiotics
have always been considered as potential human health risk
factors, over the last two decades antibiotic excretion by livestock
has come to be considered one of the major contributors to
environmental antibiotic resistance [6]. Indirect measurements
suggest that 40–90% of administered antibiotics are eliminated
from animal body via feces or urine, excreted either as the parent
compound or as metabolites [7]. Excreted antibiotics can persist in
the environment and, even at very low concentrations, can cause
emergence of antibiotic resistance in soil microorganisms, leading
to dissemination of antibiotic resistance to humans and animals
[8–11]. Therefore, reliable quantification of antibiotics excreted in
feces and urine is needed assess the environmental impact of the
livestock industry. The microbial- or immuno-assays used to test
milk and meat may yield false positive identification in manures
due to matrix interferences [12], often fail to differentiate a parent
compound from its metabolite(s) [13], suffer low detection
sensitivity [14–16], and provide only semi-quantitative results
[17]. Hence, there is a need for both qualitative and quantitative
analytical methods to better assess the fate and impact of
antibiotics in animal products, manure, and manure-impacted
environments.
PLOS ONE | www.plosone.org 1 November 2014 | Volume 9 | Issue 11 | e112343

Common uses of antibiotics on dairy farms are 1) dry cow
therapy (prophylactic, at the end of lactation) and 2) therapeutic
treatment of intramammary and other infections. In the United
States, 90% of dairy farms used dry cow therapy in all cows, and
cephapirin was the most frequently used compound [18]. In
lactating cows, following intramammary infusion of cephapirin,
60% of the administered dose was estimated to be excreted in
milk, indicating that the remaining 40% was either eliminated in
urine and feces or degraded [3]. The timing of excretion following
dry cow therapy is unknown, however, and may differ because
these dry cows are not milked after the intramammary infusion.
In early metabolism studies, cephapirin was usually quantified
using microbial inhibition immunoassay methods [1,19]. These
early methods were replaced by chromatographic methods
coupled with UV-Vis detectors, pulsed amperometric detectors,
mass spectrometry, or tandem mass spectrometry [20–23]. Most of
these methods were developed to quantify cephapirin in milk and
tissues, which have much higher concentrations of the antibiotic
than feces and urine. Also, there are more matrix interferences
from feces and urine. Therefore, established extraction, cleanup,
and analytical methods for milk and tissues may not be suitable for
quantification of trace amounts of cephapirin in feces and urine.
Continuous technological advancement of liquid chromatogra-
phy-mass spectrometry has led to the development of new
strategies to qualify and quantify antibiotics with improved
selectivity and sensitivity, and therefore, tandem mass spectrom-
etry has been preferred to other detection techniques [24–27].
High performance liquid chromatography is increasingly replaced
with ultra high performance liquid chromatography (UPLC) to
improve the resolution of chromatogram while reducing elution
time and solvent consumption [28,29].
Extraction of the analyte from sample and clean-up of sample
extracts are the keys to the sensitivity of any method involving
chromatography and mass spectrometry. Extractants commonly
used include acetonitrile (ACN) and methanol (MeOH), separately
or combined, with or without addition of buffers
[20,22,23,26,28,30]. Extraction is always followed by clean-up
steps. Solid phase extraction (SPE) using cartridges is more
popular than other clean-up approaches such as sample volume
reduction, filtration, and dispersive SPE [20,26,28,30]. The
primary goal of this project was to develop an extraction, clean-
up, and UPLC-MS/MS analytical method to qualify and quantify
trace levels of cephapirin in bovine urine and feces. Another goal
of this study was to apply the developed method to capture the
temporal pattern of cephapirin excretion in dairy cows following
intramammary infusion of cephapirin.
Materials and Methods
Chemicals and Reagents
Cephapirin standard was obtained from Sigma (Sigma, St.
Louis, MO). Analytical grade monosodium phosphate, monohy-
drate, disodium phosphate, heptahydrate, and sodium hydroxide
(1 M), and HPLC grade MeOH, ACN, and formic acid [13] were
obtained from Fisher (Pittsburgh, PA).
A stock solution of cephapirin was prepared at 100 mg mL21
by
dissolving cephapirin in ultra pure water from the Milli-Q system
(Millipore, Billerica, MA) and stored at 280uC. Intermediate stock
solutions were prepared at 10 mg mL21
by diluting stock solution
in ultra pure water. Working solutions for instrument calibration
standards and spike experiments were prepared by diluting
intermediate stock solution in MeOH. A 500 mM stock phosphate
buffer was prepared by dissolving 0.78 g monosodium phosphate
monohydrate and 65.48 g disodium phosphate heptahydrate in
500 mL water, and the pH was adjusted to 8.5 using sodium
hydroxide (1 M).
Instrumentation
Solid phase extraction vacuum manifold and OASIS HLB
(hydrophilic-lipophilic-balanced) plus short cartridge (250 mg
sorbent), used for sample clean-up, were obtained from Agilent
(Lexington, MA) and Waters (Milford, MA), respectively. Analyte
separation and quantification were performed using Agilent 1290
UPLC coupled with Agilent 6490 Triple Quad tandem mass
spectrometry (Agilent, Santa Clara, CA). Analytical (Zorbax
Extend C18 analytical column; 4.6650 mm, 5 mm particle size)
and guard columns (Zoebax Extend C18 guard column;
4.6612 mm, 5 mm particle size) were purchased from Agilent.
Animal Experiment and Sample Collection
An experiment was conducted with dairy cows to collect
samples for method development and validation and to monitor
the pattern of cephapirin excretion in dairy cows following
intramammary infusion. All procedures for this study were
approved by the Virginia Tech Institutional Animal Care and
Use Committee (IACUC protocol: 12-184-DASC). The treatment
was dry cow therapy using cephapirin benzathine (TOMOR-
ROW; Boehringer Ingelheim Vetmedica, Inc., ST. Joseph, MO).
Three end-of-lactation Holstein dairy cows were selected for the
study, housed in a individual tie stalls (1.2562.25 m), and offered
free choice water and ad libitum total mixed ration throughout the
study. On day 1 cows were fitted with urinary catheters to allow
separate collection of feces and urine. After 24 h of acclimation to
the barn and catheters, the cows were infused with 300 mg
cephapirin per quarter intramammary once, per manufacturer
instructions.
Blank fecal and urine samples were collected from dairy cows
before cephapirin infusion and used for spike recovery experi-
ments. Post-treatment fecal and urine samples were collected at 4,
6, and 8 h post-treatment and used to evaluate applicability of the
method. To monitor the extended pattern of cephapirin excretion,
daily fecal and urine samples were collected for 5 days,
subsampled from excreta accumulated over each 24 h period.
Fecal and urine samples from each cow were analyzed separately.
Extraction and Clean-up
Fecal and urine samples were extracted using 50 mM
phosphate buffer in MeOH and water with final MeOH
concentration of 50%. Feces (1 g wet) or urine (1 mL) was
weighed or pipetted into 50 mL polypropylene centrifuge tubes
and 0.5 mL of phosphate buffer (500 mM; pH 8.5), 2 mL (for
feces) or 1 mL (urine) of water, and 2.5 mL of MeOH were added
sequentially to achieve a final concentration of 50 mM phosphate
buffer and 50% MeOH(v/v). After adding the extractants, tubes
were mixed (Vortex mixer) for 10 s and sonicated at 35uC for
15 min. After the sonication, the tubes were shaken on a
horizontal shaker (Reciprocal Shaker; Model E6000; Eberbach
Corp., Ann Arbor, MI) at a speed of 260 osc min21
with
horizontal stroke of 38 mm for 30 min at ambient temperature
(,20uC). After shaking, the samples were then centrifuged at
30,0006g for 15 min at 4uC. All supernatants were decanted into
glass tubes, and diluted to 50 mL using 50 mM phosphate buffer.
Tubes were inverted for 4–5 times to achieve homogeneity of
sample extract and phosphate buffer.
Solid phase extraction was used to further remove matrix
interference from each extract. For the SPE, OASIS HLB
(hydrophilic-lipophilic-balanced) plus short cartridge (250 mg
sorbent; Waters, Milford, MA) and 20 port SPE vacuum manifold
UPLC-MS/MS Quantification of Cephapirin in Bovine Feces and Urine

(Agilent, Lexington, MA) were used. A 20 mL reservoir was
mounted above each cartridge to accommodate sample extract
and chemicals for conditioning, washing, and elution. Cartridges
were conditioned with MeOH, water, and phosphate buffer
sequentially by applying vacuum (Table 1). Sample extracts were
loaded onto conditioned cartridges and vacuum was controlled to
achieve a flow rate of ,3 mL per min. Following sample loading,
cartridges were washed with phosphate buffer and water
sequentially (Table 1). After the washing step, vacuum was applied
to draw all liquid out of the cartridges and the cartridges were
allowed to dry for 4 min. Next cephapirin was eluted sequentially
with 3 mL MeOH and 3 mL ACN into the same tube (Table 1).
Eluted extracts were mixed using a Vortex mixer (30 s) and by
inverting (4–5 times). An aliquot of 1 mL eluted extract was
transferred to a 10 mL glass tube and dried under a gentle stream
of nitrogen gas at 35uC using a Zipvap 20 evaporator (Glas-Col,
Terre Haute, IN). Then 1 mL of MeOH:water (30:70, v/v) with
0.1% FA was added to each tube to completely dissolve the dried
cephapirin residue. The 1 mL solution in each tube was mixed
(Vortex mixer) for 30 s, and filtered through 0.2 mm PVDF
syringe filter (Fisher, Pittsburgh, PA) into 1.5 mL amber glass
HPLC vials for the UPLC-MS/MS analysis.
UPLC-MS/MS operating Conditions and Cephapirin
Qualification and Quantification
Cephapirin was analyzed using Agilent 1290 UPLC coupled
with Agilent 6490 Triple Quad tandem mass spectrometry
(Agilent, Santa Clara, CA, USA). Electrospray negative ionization
in multiple-reaction monitoring mode was used. Zorbax Extend
C18 analytical column (4.6650 mm, 5 mm particle size, Agilent,
Santa Clara, CA, USA) coupled with Zorbax Extend C18 guard
column (4.6612 mm, 5 mm particle size, Agilent, Santa Clara,
CA, USA) was used for chromatographic separation. Sampler and
column compartments were kept at 8 and 40uC, respectively. The
injection volume was 10 mL. A gradient elution program
consisting of two mobile phases (mobile phase A: 0.1% FA in
water; mobile phase B: 0.1% FA in MeOH; Table 2) was used at a
flow rate of 0.5 mL min21
. Mass spectrometry parameters are
listed in Table 3. Cephapirin in positive and spiked samples were
qualified by comparing LC-MS/MS spectra of samples with those
of cephapirin standards. Accepted variation in mass to charge ratio
was 10%, and acceptable variation was set as 20% for the ratio of
qualifier and quantifier ions. Cephapirin concentration in tested
samples was quantified using the calibration curve of seven matrix-
matched cephapirin standards (0.7, 1, 2, 4, 5, 10, and 20 mg L21
matrix solution). Matrix-match standards were prepared using the
SPE cleaned-up extracts of blank feces or urine samples.
Method Validation
Limit of quantification (LOQ) and limit of detection (LOD)
were determined using the equations: LOQ = 10(SD/S) and
LOD = 3.3(SD/S) [31], where S is the slope of a calibration
curve of seven matrix-match standards and the SD is the standard
deviation of responses from seven replicates of the lowest matrix-
match standard. Matrix effect (%) was calculated using the
equation: [{(peak area of cephapirin in matrix/peak area of
cephapirin in solvent)21}6100].
Linearity of the instrument was checked by analyzing nine
cephapirin standards (1–500 mg L21
) prepared in MeOH:ACN
(50:50, v/v) with each concentration injected three times. A
calibration curve was constructed by plotting peak areas for the
standards against its concentrations. The calibration equation and
correlation coefficient from the regression analysis were used to
validate linearity.
Spike recovery tests were performed by spiking matrix-match
standards to 1 g feces (wet weight) or 1 mL urine before extraction
(pre-extraction) and in extracts (post-extraction). Three different
spike concentrations were selected based on the LOQ values for
cephapirin in feces and urine matrix [31]. For pre-extraction spike
tests, 1 g feces or 1 mL urine was spiked with 1 mL of spike
solutions (prepared in MeOH) to achieve concentrations of 2.5, 5,
and 10 LOQ and was equilibrated for 2 min before extractant was
added to each spiked sample. The extraction, cleanup, and
analysis procedures were as described in the previous sections. For
post-extraction recovery tests, feces or urine extracts were spiked at
concentrations of 2.5, 5, and 10 LOQ. Spiked extracts were
equilibrated for 2 min and mixed using a Vortex mixer to achieve
homogenous mixing followed by clean-up and analysis using the
procedures described previously.
Intra-day precision was evaluated by analyzing, at different
times within one day, six replicates of cephapirin-spiked blank
samples (feces or urine) at three concentrations (2.5, 5, and 10
LOQ). Inter-day precision was assessed on four different days by
preparing and analyzing three replicates of cephapirin-spiked
blank feces or urine at 2.5, 5, and 10 times of their respective LOQ
values. Matrix effect of feces and urine was evaluated by
comparing the peak area response of seven cephapirin standards
dissolved in MeOH:water (30:70, v/v, 0.1% FA) with the peak
area response of those dissolved in blank fecal and urine extracts at
a concentration range of 1–50 mg L21
.
All calibration standards were dried under N2 and redissolved in
1 mL MeOH:water (30:70, v/v, 0.1% FA) using the same
procedures as for the SPE cleaned-up sample extracts in order
to eliminate variation due to any loss of cephapirin during the N2
drying process.
Table 1. Solid phase extraction conditions.
Step Solvent Volume, mL Destination
1 Conditioning Methanol 3 Discard
2 Conditioning Ultra pure water 3 Discard
3 Conditioning Phosphate buffer (50 mM, pH 8.5) 3 Discard
4 Washing1
Phosphate buffer (50 mM, pH 8.5) 2 Discard
5 Washing1
Ultra pure water 2 Discard
6 Elution1
Methanol 3 Collect
7 Elution1
Acetonitrile 3 Collect
1
Flow rate was ,3 mL/min for washing and elution steps.
doi:10.1371/journal.pone.0112343.t001

Statistical Analysis
Recovery values and cephapirin concentrations in samples are
reported as arithmetic means of triplicates with standard deviation
calculated using Microsoft Excel. Linear regression analysis was
performed in Microsoft Excel to test linearity of calibration curve.
Precision was estimated as residual standard deviation using
Microsoft Excel.
Excretion data were analyzed using the GLIMMIX procedure
in SAS (SAS Inst. Inc., Cary, NC) with cow (n = 3) as the
experimental unit. The statistical model included day as fixed
effect, cow as a random variable, and pre-treatment data as a
covariate. Orthogonal polynomial contrasts were used to test the
linear, quadratic, and cubic effects of day.
Results and Discussion
Optimization of Extraction
The extraction step was optimized by testing different extract-
ants used by others to extract cephapirin or cephalosporins from
milk or biological fluid samples. Phosphate buffer at pH = 8.5 was
tested to extract cephapirin from feces because previous studies
have shown that high pH phosphate buffers (pH 8.5 to10)
recovered cephapirin completely from milk, while a low pH
phosphate buffer (pH 3.2) was inefficient in extracting cephapirin
from egg [28,30]. In our experiment, phosphate buffer (500 mM;
pH 8.5) alone recovered ,20% of cephapirin from feces when
different sample to extractant weight to volume ratios (1:1, 1:5,
and 1:10) were tested. A mixture of methanol and 50 mM
phosphate buffer (pH 8.5) at 50% (v/v) as extractant enhanced the
recovery of cephapirin from feces (40%).
Previous research has shown that organic solvents such as
acetonitrile, methanol, or their combination could recover .70%
cephapirin from milk and serum [22,23] so this approach was
evaluated. Recovery was improved to .60% when a sample was
first mixed at weight/volume ratio of 1:5 for feces and volume/
volume ratio of 1:4 for urine with a methanol/50 mM phosphate
buffer (pH 8.5) mixture (50%, v/v), and then sonicated at 35uC for
15 min followed by additional shaking for 30 min on a horizontal
shaker at 260 osc min21
with horizontal stroke of 38 mm.
Before the SPE clean-up step, 5 mL extract was diluted with
phosphate buffer (50 mM) to 50 mL to bring the final concentra-
tion of MeOH in the diluted extract to below 10%, because
organic solvent above this concentration was reported to elute
cephalosporin antibiotics from SPE cartridges [32].
Optimization of SPE Clean-up Step
The OASIS HLB cartridge (Waters, Milford, MA) was selected
for this experiment because it is commonly used to reduce matrix
effects in milk and tissue samples during sample clean-up for
cephalosporin analysis [26,30,32]. The SPE clean-up step was
optimized by testing different sequences and composition of
conditioning, and elution solvents. The optimization process
started with cephapirin-spiked water acidified with different
volumes (50, 100, and 200 mL) of 2 M HCl with cartridge
conditioning (with 2 mL ACN and 2 mL water), washing (with
3 mL water and 3 mL 3% ACN), and elution (3 mL ACN and
3 mL acetone) solvents fixed. With increasing volume of 2 M HCl,
cephapirin recovery gradually decreased from 40 to 15%. It was
determined that 50 mL of 2 M HCl was optimum for acidification.
Table 2. Chromatographic conditions.
Mobile phase
Time (min) A1
, % B2
, %
0 70 30
6 5 95
7.5 70 30
12 70 30
1
A: 0.1% formic acid in water.
2
B: 0.1% formic acid in methanol.
Table 3. MS/MS operating conditions.
Parameters
Ionization mode Electrospray negative ionization
Data collection Multiple-reaction monitoring (MRM)
Nebulizer gas flow 16 L/min
Capillary voltage 3000 V
Fragmentation voltage 380 V
Collision energy 15 V
Ion source temperature 250uC
Precursor ion (m/z) 424
Qualifier ion (m/z) 181
Quantifer ion (m/z) 292

After determining the optimum acidification step for cephapirin
spiked-water, different elution solvents were tested. Cephapirin
recovery was ,50% when cephapirin was eluted from the HLB
cartridge using ACN and acetone mixed together or sequentially
in different proportions (ACN:acetone, 50:50 or 80:20, v/v; 3 or
4 mL ACN followed by 3 or 1 mL acetone). Maximum recovery
of .60% was later achieved when non-acidified cephapirin
spiked-water was loaded onto HLB cartridge pre-conditioned with
MeOH, water, and phosphate buffer (Table 1), followed by
washing the cephapirin loaded cartridge with phosphate buffer
(Table 1) and water, and sequentially eluting cephapirin off the
cartridge using 3 mL MeOH and 3 mL ACN (Table 1). This
optimized SPE clean-up approach was applied to clean up of
cephapirin-spiked blank fecal and urine samples and resulted in
recoveries of .60% and .80%, respectively (Table 4).
Method Validation and Application
The optimized extraction and clean-up steps were validated for
quantification of cephapirin in cephapirin-spiked blank bovine
feces and urine and those from animals administrated with
cephapirin.
Figure 1 and 2 show the chromatograms of blank feces and
urine from a dairy cow not treated with cephapirin, cephapirin-
spiked (at 10LOQ) blank feces and urine, feces and urine collected
from a dairy cow 4 h after it was treated with cephapirin, 50 mg
L21
cephapirin standard, and 20 mg L21
cephapirin drug
dissolved in solvent (this last being the form of drug administered
to the cow).
As shown in Figure 3, both feces and urine had matrix effects,
with larger slope values for calibration curves prepared from
calibration standards dissolved in LC mobile phase solvent as
compared to that for cephapirin standards dissolved in fecal or
urine matrix. The matrix effects for feces and urine were 229 and
220%, respectively (Table S1). Negative matrix effect indicated a
suppression of response. Integration of peak area accounted for
double peak and double peak area was used to plot all calibration
curves or spiked-blank fecal samples. Calibration standards were
prepared fresh on the day of analysis (although the standard
prepared in fecal matrix at 1 mg L21
was stable for one month at
220uC; data not shown). The limit of quantification for
cephapirin in cephapirin-spiked blank bovine feces and urine
was 4.02 mg kg21
(wet weight), and 0.96 mg L21
, respectively.
To our knowledge, this is the first study where LOQ was
determined for cephapirin in bovine feces and urine. In this
experiment matrix-match calibration standards were used to
reduce the effect of matrix during cephapirin quantification
(Figure 3a, b). As shown in Figure 4, the instrument response was
linearly correlated (r2
= 0.9990) with cephapirin concentration
within the range of 1 to 500 mg L21
(Table S2). All the standard
curves used for cephapirin quantification in samples were within
this range (Figure 4).
The accuracy of the method was evaluated by spike recovery
tests using blank feces and urine (samples from cows before they
were treated with cephapirin). The spike recovery tests were
divided into two segments: pre- and post- extraction. Pre-
extraction recovery tests (cephapirin spiked in blank feces or urine
before extraction) were used to assess the efficiency of the entire
method including extraction, clean-up, and quantification steps.
The efficiency of the steps from SPE clean-up to quantification
was evaluated using post-extraction recovery tests by spiking
cephapirin in the extracts of blank feces or urine. Pre-extraction
recovery of cephapirin in blank feces and urine samples ranged
from 64 to 73% and 81 to 84%, respectively (Table 4; Table S3).
Post-extraction recoveries of cephapirin were higher at 95 to 100%
and 90 to103%, respectively, for feces and urine (Table 4; Table
S3).
The precision of the method was evaluated in terms of
repeatability (intra-day precision, % RSD) and reproducibility
(inter-day precision, % RSD). The repeatability for cephapirin in
spiked blank feces and urine was 7.99% and 3.07%, respectively
(Table 4; Table S3), which are within the acceptable method
Table 4. Method validation data.
Feces Urine
LOQ1
(mg kg21
or mg L21
) 4.02 0.96
LOD1
(mg kg21
or mg L21
) 1.33 0.32
Pre-extraction spike recovery2
(%)
Spike level
2.56LOQ 7364.7 8163.5
56LOQ 6961.9 8261.3
106LOQ 6462.5 8462.7
Post-extraction spike recovery2
(%)
Spike level
2.56LOQ 9662.8 9065.9
56LOQ 9564.8 10361.2
106LOQ 10060.3 9464.6
Precision (%RSD)
Intra-day3
(n = 18) 7.99 3.07
Inter-day4
(n = 36) 8.18 9.59
1
LOQ and LOD: mg kg21
wet feces or mg L21
urine.
2
Recoveries are given as mean6standard deviation (n = 3).
3
Intra-day variation was calculated using six replicates of three spike concentrations.
4
Inter-day variation was calculated using three replicates of three spike concentrations for four days.

repeatability guidelines set by FDA [33]. The reproducibility
values were 8.18 and 9.59% for spiked blank feces and urine,
respectively (Table 4; Table S3).
Application of the developed analytical approach was evaluated
by analyzing feces and urine collected from dairy cows at 4, 6, and
8 h following intramammary infusion of cephapirin-based antibi-
otic. Cephapirin was detected at 2.04 and 2.12 mg kg21
(wet
weight) in the feces collected at 4 and 6 h post cephapirin
administration, but was below the detection limit in feces collected
at 8 h post cephapirin administration (Table 5; Table S4).
Cephapirin concentrations were 133 and 480 mg L21
in the urine
samples collected at 4 and 8 h post cephapirin administration
(Table 5; Table S4). Reported cephapirin concentrations in feces
and urine were not normalized with their respective recoveries
shown in Table 4.
In spite of its primary use as a veterinary antibiotic, cephapirin
may cause development of resistance to antibiotics critical for
human health, because exposure to one antibiotic compound can
cause bacteria to develop resistance to other antibiotics [34,35]. In
addition, cephapirin-induced antibiotic resistance genes in the
animal gut, manure, or soil may be acquired by bacteria
pathogenic to humans. The application of this method to feces
and urine from antibiotic-treated cattle will aid in efforts to identify
environmental practices (manure treatment, runoff control mea-
sures) to reduce loading of antibiotics to the environment.
Therefore, the development of this method and improved methods
to measure other antibiotics in manure has direct implications for
public health.
Temporal Pattern of Cephapirin Excretion
Cephapirin was not detected in the 24 h cumulative samples of
feces collected for 5 days following intramammary infusion of
cephapirin. This is because cephapirin benzathine was synthesized
with the sole focus on intramammary infusion. During the
synthesis of any drug that is intended for local therapy, a goal is
that the drug should not reach non-target areas (in this case, the
digestive tract). Therefore, local intramammary infusion ideally
would not lead to significant excretion in the feces. Also, any
cephapirin entering the digestive tract would be subject to
Figure 1. UPLC-MS/MS MRM chromatograms for cephapirin (a) in a blank fecal sample (feces from a dairy cow before treated with
cephapirin), (b) in blank feces spiked with cephapirin at 10LOQ, (c) in a fecal sample collected from a dairy cow 4 h after cephapirin
was administered, (d) a cephapirin standard dissolved in MeOH:water (30:70, v/v, 0.1% formic acid) (20 mg L21
).
doi:10.1371/journal.pone.0112343.g001

degradation by intestinal bacteria. The enzyme b-lactamase,
active on cephalosporins, was detected in bacteria isolated from
the digestive tract of cows not exposed to antibiotics, and
cephalosporins (ceftiofur and ceftriaxone) were degraded in the
presence of those bacterial isolates [36]. Cephalosporin (ceftiofur)
was degraded almost completely within 8 h when incubated at
room temperature in the presence of bovine feces [37]. The critical
role of fecal microbes in degrading cephalosporin was confirmed
by low-extent of degradation (,40%) of ceftiofur in the presence
of sterile feces even after 48 h of incubation [37].
Urine was the primary route of cephapirin excretion in lactating
cows as has been observed in other animals including humans
[38–41]. Cephapirin excretion in urine was influenced by day
(P = 0.03) with peak excretion (2.69 mg) on day 1 following
intramammary infusion (Figure 5). After the peak on day 1,
cephapirin excretion decreased sharply on day 2 (0.19 mg) and did
not change for rest of the study (0.19, 0.08, and 0.17 mg on day 3,
4, and 5, respectively; Table S5) reflecting quadratic pattern of
urinary cephapirin excretion (Quadratic: P = 0.03; Figure 5).
Drug molecules are transported from milk to blood primarily
via passive diffusion [42]. Initial peak urinary excretion of
cephapirin on day 1 post-treatment was likely due to faster
diffusion of cephapirin along high concentration gradient from its
high concentration in the milk to low concentration in the blood.
Passive diffusion of drugs across the milk-blood barrier requires
wide distribution of the drug throughout the udder. This is
regulated by three characteristics of the drug 1) lipid solubility, 2)
rate and extent of ionization, and 3) milk protein or tissue binding
[43]. Low lipid solubility of cephapirin may explain low absorption
of cephapirin in this study, but does not explain the temporal
variation in urinary excretion as any change in cephapirin
solubility is not expected over the time. It would be expected
that milk pH remained relatively stable during these experiments
because cows did not have clinical mastitis and were not subject to
dietary changes. Therefore, temporal variation in cephapirin
excretion cannot be attributed to the change in ionization rate of
cephapirin due to pH variation, so binding to tissue or milk
proteins is the likely explanation. Drugs or antibiotics diffuse
across the milk-blood barrier only in the unbound form. When
antibiotics bind to udder tissue or milk protein, only a small
proportion of the administered dose is available for absorption
[44–46]. Peak excretion of cephapirin in urine within 24 h of
intramammary infusion of cephapirin suggests saturation of
binding sites immediately post-treatment due to the initial high
concentration of cephapirin, leaving a major proportion of the
administered dose available for absorption. Similarly, tetracycline
absorption from bovine udders gradually decreased with time
following infusion, an observation attributed to saturation of
antibiotic binding sites [42].
After a transitory increase on day 1, the sharp decrease in
cephapirin excretion on day 2 was likely due to increased binding
of cephapirin to tissue protein because the extent of drug binding
to protein increases with the decrease in drug concentration [47].
Also, with time, conversion of cephapirin to its major metabolite
desacetyl cephapirin is likely. Within 24 h of intramammary
infusion of cephapirin in lactating cows .50% of infused
cephapirin was converted to desacetyl cephapirin [3,48].
When expressed as a proportion of total dose, only 0.22% was
excreted on day 1 after intramammary infusion. On following days
excretion ranged from 0.01 to 0.02% of total cephapirin
administered. Cumulative excretion of cephapirin for 5 days
post-treatment was 0.28%. The excretion of only a very small
proportion of total intramammary dose was likely due to low
solubility of cephapirin leading to limited distribution throughout
the udder and subsequently poor absorption [44,49]. This low
solubility is intentional to increase the duration of therapy during
the dry period (,45–60 days). Milk is mildly acidic (pH 6.4–7) and
,99% of cephapirin is ionized at pH 6.4 [44,50]. Therefore, only
1% would be available as nonionized (absorbable) form. Also each
nonionized molecule will not necessarily be absorbed because
binding of nonionized cephapirin to milk protein or tissue would
limit passive diffusion of cephapirin. The large size of the
cephapirin benzathine molecule is another factor that might have
contributed to poor absorption of cephapirin across the milk-blood
barrier. Similarly, penicillin benzathine was poorly absorbed in the
bovine udder [51].
Cephapirin excretion expressed as a proportion of total dose
can provide a basis of indirect comparative risk assessment of
antibiotic loading to the environment, by comparison with
excretion of other cephalosporins or other classes of antibiotics
Figure 2. UPLC-MS/MS MRM chromatograms for cephapirin (a) in a blank urine sample (urine from a dairy cow not treated with
cephapirin), (b) in blank urine spiked with cephapirin at 10LOQ, (c) in a urine sample collected from a dairy cow 4 h after
cephapirin was administrated.

following systemic administration in animals and human. When
administered via intravenous (IV) and intramuscular (IM) routes at
950 mg/subject in humans, the proportion of administered
cephapirin excreted in urine was 48% after 6 h of IV and 43%
after 24 h of IM [39]. A similar extent of cephapirin excretion was
observed in humans receiving 1000 mg of cephapirin by the IV
route [38]. Urinary excretion of cephapirin varied in small animals
with the route of administration. In mice, 20% of the total
cephapirin dose was excreted in urine within 24 h of subcutaneous
administration (30 mg/kg), but the rate and extent of excretion
(32% of total dose within 8 h of dosing) was higher in dogs
administered with cephapirin at 30 mg/kg by the IV route [38].
Following an oral dose of cephradine, another cephalosporin, 84
and 90% of the total dose was excreted in the urine of mice and
rats [41]. In dogs, within 7 h of an oral dose (50 mg/kg) of
cephradine, .50% of the administered dose was excreted in feces
Figure 3. Calibration curves of standards prepared (a) in blank fecal matrix and solvent [MeOH:water (30:70, v/v, 0.1% formic
acid)]; (b) in blank urine matrix and solvent.

and urine. In pigs, fecal and urinary excretion of ceftiofur ranged
from 81 to 95% of total administered when IM dose varied
between 3.08 and 6.76 mg/kg body weight [52]. Ceftiofur was
almost completely (92.5% in urine and 6.5% in feces) excreted by
sheep administered with 5 IM doses at 2.2 mg/kg body weight
[53]. These cephalosporin formulations are designed to be
absorbed for distribution to target tissues. In contrast, the
cephapirin formulation used in this study is designed to be locally
active to increase the duration of therapy during the dry period
(,45 to 60 days). Therefore, there is less chance that a higher
proportion of intramammary dose would be excreted.
Even though only a small proportion of intramammary
cephapirin dose was excreted in urine, the concentration range
of urinary cephapirin (5.94 to 240 mg L21
) in this study indicated
the potential of excreted cephapirin residue in exerting selection
pressure on environmental microbial communities. Cephapirin is
primarily used as dry cow therapy in dairy cows, and on most
dairy farms in the US cows are housed on pasture or drylots
during the dry period [54]. Therefore, cephapirin excreted in
urine will enter the soil and its concentration in soil will depend on
the distribution and transportation of cephapirin in the soil. Dose-
response relationships are not established to know what concen-
tration of which antibiotic will cause antibiotic resistance
development in various bacterial species under specific environ-
mental condition. Because of these uncertainties, all that can be
discussed is the potential of excreted cephapirin concentration
Figure 4. Linearity of standard curve for cephapirin standards dissolved in MeOH:water (30:70, v/v, 0.1% formic acid) at
concentrations ranging from 1 to 500 mg L21
.
Table 5. Quantification of cephapirin in feces and urine collected from dairy cows 4, 6, and 8 hours after they were treated with
cephapirin.
Hours after dairy cows
were treated with cephapirin
Concentration1
(mg kg21
or L21
)
Feces 4 2.0460.302
6 2.1260.092
8 ND3
Urine 4 13361.35
8 480617.4
1
Results are given as mean6standard deviation (n = 3).
2
Cephapirin concentration in 4 and 6 h fecal samples are . LOD but , LOQ.
3
ND = Below LOD.

with respect to minimum inhibitory concentration (MIC) of
cephapirin estimated in laboratory conditions.
Reported MIC of cephapirin for several bacterial species (e.g.
Staphylococcus aureus, Streptococcus uberis, Streptococcus dysga-
lactiae, Escherichia coli, Pseudomonas aeruginosa, Proteus mir-
abilis) ranged from 30 to 64,000 mg L21
when inoculum size
varied from 104
to 107
cells mL21
[55–60]. Our discussion will
consider reported MIC values of cephapirin, but it should be kept
in mind that most of these MIC values were estimated by in vitro
susceptibility tests using nutrient rich pure growth media with
fixed initial inoculum size, and MIC values vary with several
factors including inoculum size and the substrates present in the
media [55,58,61]. Since there are no established MIC values of
cephapirin for soil matrices, we will discuss the potential of
excreted urinary cephapirin in antibiotic resistance development
considering two scenarios: cephapirin concentration 1) higher than
MIC and 2) lower than MIC. The range of urinary cephapirin
concentration in this study (5.94 to 240 mg L21
) overlaps with
reported MIC values (30 to 64,000 mg L21
) and exceeds the MIC
values of cephapirin for some bacterial species. If the soil bacterial
community is exposed to a concentration of cephapirin that is
higher than MIC, resistant species of bacteria will grow due to
selection pressure and contribute to the environmental antibiotic
resistome. The most likely scenario is that the concentration of
cephapirin in soil will be much lower than the urinary
concentration we observed in this study due to degradation,
distribution in larger area, and adsorption to soil particles.
Antibiotics are detected in the environmental samples at parts
per trillion (ppt) or parts per billion (ppb) levels [27]. If cephapirin
concentration in soil is far below MIC, it can still contribute to
antibiotic resistance development in soil bacterial community
[62,63]. Antibiotic concentrations ranging from J to 1/230 of
MIC for susceptible strains exerted selection pressure on certain
bacterial communities leading to increased antibiotic resistance,
and this increase was attributed not only to enrichment and
maintenance of pre-existing resistance but also to the de novo
selection of new resistant traits [64].
Although intramammary infusion of cephapirin is used on .
90% of dairy farms in the US, the contribution of this
management practice to the environmental pool of antibiotic
residue is likely lower than when the same drug is used
therapeutically. If manure treatment methods are developed that
are effective in degrading antibiotics, manure from cows on day 1
following dry cow therapy would be the priority for segregation
and treatment. However, manure from days 2 through 5 might
also need to be treated or stored for longer duration to ensure
complete degradation of cephapirin, because antibiotics even at
very low sub-MIC concentrations can exert selection pressure.
The results of this study will help to develop efficient management
strategies to reduce the development of antibiotic resistance in the
environment.
Conclusions
A method was developed and validated for qualification and
quantification of cephapirin in bovine feces and urine, including
extraction and clean-up, coupled with UPLC-MS/MS. This
method is appropriate both qualitatively and quantitatively for
detection of cephapirin in feces and urine with very low LOQ.
This method can be applied to qualify and quantify cephapirin in
bovine feces and urine with high accuracy. It allows measurement
of trace amounts of cephapirin typical of those present in feces and
urine from treated cattle, and thus will help assess environmental
loading of antibiotics from the livestock industry. Urinary
excretion of cephapirin followed a quadratic pattern with peak
excretion on day 1 post-treatment followed by a sharp decrease on
day 2. Excretion of only a minor proportion of total cephapirin
dose indicates that the environmental loading of cephapirin due to
intramammary cephapirin use in dairy cows is less compared to
therapeutic use of other antibiotics in human and animals.
Figure 5. Daily urinary excretion of cephapirin (mg) in dairy cows administered with cephapirin (300 mg/quarter) via
intramammary route. Data is represented as least square means with error bars as standard errors. Symbol * indicates that day 1 is significantly
(P,0.05) different from all other days.

Supporting Information
Table S1 Effect of feces or urine matrix on cephapirin
quantification.
(PDF)
Table S2 Linear correlation between cephapirin con-
centration and instrument response.
(PDF)
Table S3 Recovery of cephapirin spiked in feces or
urine or in their extracts.
(PDF)
Table S4 Cephapirin concentration in feces and urine
collected from dairy cows treated with cephapirin.
(PDF)
Table S5 Daily urinary excretion of cephapirin in dairy
cows following intramammary infusion of cephapirin.
(PDF)
Acknowledgments
This activity was funded, in part, with an integrated, internal competitive
grant from VAES, VCE, and the College of Agriculture and Life Sciences
at Virginia Tech. This article was supported by Virginia Tech’s Open
Access Subvention Fund.
Author Contributions
Conceived and designed the experiments: KFK PR KX CS. Performed the
experiments: PR. Analyzed the data: PR. Contributed reagents/materials/
analysis tools: KFK KX. Contributed to the writing of the manuscript: PR
KFK KX CS.
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