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Method Development and Validation: Solid Phase Extraction-Ultra Performance Liquid Chromatography-Tandem Mass Spectrometry Quantification of Pirlimycin in Bovine Feces and Urine Partha ray and Katharine F. KnowltonVirginia Polytechnic Institute and State University, Department of Dairy Science, Blacksburg, VA 24061Chao Shang and Kang XiaVirginia Polytechnic Institute and State University, Department of Crop and Soil Environmental Sciences, Blacksburg, VA 24061

Received February 20, 2014. Accepted by JB May 26, 2014.Corresponding author’s e-mail: drray@vt.eduDOI: 10.5740/jaoacint.14-040

VETERINARY DRUG RESIDUES

Pirlimycin, a lincosamide antibiotic, is one of the

most commonly used antibiotics for the treatment

of mastitis in dairy cows. Assessment of pirlimycin

loading to the environment via fecal and urinary

excretion is critical to develop efficient management strategies to reduce environmental pollution by the

livestock industry. Therefore, the aim of this study

was to develop and validate an analytical method

to identify and quantify pirlimycin in bovine feces

and urine. Samples were extracted with methanol–

phosphate buffer and cleaned up by SPE before

analysis for pirlimycin using UPLC-MS/MS. This

method was sensitive (LOQ 1.47 ng/g wet feces,

0.90 ng/mL urine), accurate (recovery, 80–108%),

and precise (repeatability, 2.3–13%; reproducibility,

2.3–14%) for both bovine feces and urine. With the

application of this method to samples collected in

the first 10 h and then every 24 h for 120 h following intramammary dosing (50 mg/cow; n = 3 cows),

pirlimycin was detected at 40.5–287 ng/g and

46.1–254 ng/mL in feces and urine, respectively.

This robust, sensitive, and accurate method can be

used to assess the fate and environmental impact of

antibiotics used on farms.

The use of antibiotics in food animals has been a major concern for human health because of the risk of antibiotic residues in animal food products. In addition

to this problem, association of antibiotic use in food animals and increasing emergence of antibiotic resistance has received great attention in recent years. Following administration, antibiotics are distributed in different tissues or secreted in milk (1–5) before elimination from the body via feces and urine. As per indirect measurements, 40–90% of administered antibiotics are eliminated from animal body via fecal and urinary excretion, either as the parent compound or as metabolites (6). In the last two decades, antibiotic excretion by livestock has come to be considered as one of the major contributors to bacterial antibiotic resistance in the environment (7). Excreted antibiotics are stable in environmental matrixes/conditions, and

even at very low concentration, antibiotics can facilitate the development of antibiotic resistance in soil microbes (8–11). Therefore, accurate and reliable quantification of antibiotics excreted in feces and urine is needed to allow assessment of the environmental impacts of the livestock industry.

The major proportion of therapeutic antibiotic use in dairy cows is for treatment of mastitis (12). Mastitis is one of the most common diseases of dairy cattle, and extended loss of production is expected if cows are not treated with antibiotics (13, 14), leading to serious negative economic impacts. Pirlimycin is one of the most commonly used antibiotics for treatment of mastitis (15). Pirlimycin is a semi-synthetic derivative of lincomycin from the lincosamide group of antibiotics. In spite of its heavy use in dairy industry, there is limited research on metabolism and residue studies for pirlimycin. In pre-approval studies using radiolabeled pirlimycin, the compound was excreted primarily in feces (24% of administered dose) and 10% of total dose was excreted in urine following intramammary infusion in lactating cows (3).

The lack of experimental data quantifying excretion is probably due to inadequate methodology to quantify pirlimycin in different matrixes. As is the case with other antibiotics, commercial kits are available as screening tools to detect pirlimycin residues in biological samples. Commercial kits are based on microbial growth inhibition and provide only semiquantitative results, with frequent false positive or false negative results (16, 17). Multiclass, multiresidue LC/MS/MS methods can replace screening kits while maintaining high throughput by monitoring a wide variety of antibiotics (18). An LC method was developed to quantify pirlimycin in human serum and urine using a UV-Vis or fluorescence detector with very low LOQ (0.1–5 ng/mL; 19). But this method requires a complicated derivatization step for sample preparation. Another LC method, developed to determine pirlimycin residue in bovine milk and liver, uses mass spectrometric detection with comparatively higher LOQ values ranging from 25 to 100 ng/g or mL, for liver tissue and milk (20). These LC methods include acetonitrile extraction of pirlimycin followed by SPE sample cleanup. These extraction, cleanup, and quantification approaches were optimized using milk, animal tissue, or human urine samples, and may not be applicable to bovine feces and urine containing a comparatively more complex matrix and much lower concentration of pirlimycin. The aim of this study was to develop an extraction, cleanup, and UPLC-MS/MS

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analytical method to identify and quantify pirlimycin in bovine feces and urine at nanogram or subnanogram concentrations.

Experimental

Chemicals and Reagents

(a) Methanol.––LC grade (Fisher Scientific, Pittsburgh, PA). (b) Acetonitrile.––LC grade (Fisher).(c) Formic acid.––LC grade (Fisher).(d) Water.––Ultra pure Milli-Q water (Millipore, Billerica,

MA).(e) Standard.––Pirlimycin standard from Toronto Research

Chemicals (Toronto, Ontario, Canada).(f) Sodium hydroxide (1 M).––Analytical grade (Fisher).(g) Monosodium phosphate, monohydrate.––Analytical

grade (Fisher). (h) Disodium phosphate, heptahydrate.––Analytical grade

(Fisher). (i) Phosphate buffer (500 mM; pH 8.5).––Buffer was

prepared by dissolving 0.78 g monosodium phosphate monohydrate and 65.48 g disodium phosphate heptahydrate in 500 mL water. The pH was adjusted to 8.5 using 1 M sodium hydroxide.

Standard Solutions

A stock solution of pirlimycin was prepared at a concentration of 100 µg/mL by dissolving pirlimycin in methanol–ultra pure water (50 + 50, v/v) and stored at –80°C. Intermediate stock solutions were prepared at a concentration of 10 µg/mL by diluting stock solution in methanol–ultra pure water (50 + 50, v/v). Working solutions for instrument calibration standards, and spike experiments were prepared by diluting intermediate stock solution in methanol.

Apparatus

(a) Sonicator.––VWR Sonicator, Model B2500A-MT (VWR, West Chester, PA).

(b) Shaker.––Reciprocal Shaker, Model E6000 (Eberbach Corp., Ann Arbor, MI).

(c) Centrifuge.––Avanti J-25 High Performance Centrifuge (Beckman Coulter, Fullerton, CA).

(d) SPE set up.––OASIS HLB (hydrophilic-lipophilic-balanced) Plus Short Cartridge (250 mg sorbent, Waters Corp., Milford, MA) fitted in 20 port SPE vacuum manifold (Agilent Technologies, Lexington, MA).

(e) Nitrogen evaporator.––Zipvap 20 evaporator (Glas-Col, Terre Haute, IN).

(f) Polyvinylidine difluoride (PVDF) filter.––PVDF syringe filter of pore size 0.2 µm (Fisher).

(g) UPLC-MS/MS system.––Agilent 1290 UPLC coupled with Agilent 6490 Triple Quad tandem mass spectrometer (Agilent Technologies, Santa Clara, CA).

(h) Analytical and guard columns.––Zorbax Extend C18 analytical column (4.6 × 50 mm, 5 µm particle size, Agilent)

coupled with Zorbax Extend C18 guard column (4.6 × 12 mm, 5 µm particle size, Agilent).

Samples

For method development, fecal and urine samples were collected from cows not treated with pirlimycin. Method applicability was tested by analyzing fecal and urine samples from cows treated with 50 mg pirlimycin intramammary twice at a 24 h interval (positive samples). Positive samples included hourly samples collected at 4, 6, and 10 h following first intramammary infusion, and daily samples collected from total daily fecal and urine output for 5 days.

Sample Preparation: Extraction and Cleanup

Fecal and urine samples were extracted using 500 mM phosphate buffer in methanol and water with final buffer and methanol concentration of 50 mM and 70%, respectively. The extraction method was optimized for 1 g wet feces and 1 mL urine. One g wet feces or 1 mL urine was weighed or pipetted into 50 mL polypropylene centrifuge tubes and 0.5 mL of 500 mM phosphate buffer (pH 8.5); 1 mL water (only for feces) and 2.5 mL methanol were added sequentially to achieve the desired concentration of phosphate buffer (50 mM) and methanol (70%). These were vortexed 10 s and sonicated 15 min at 35°C, followed by shaking 30 min on a horizontal shaker at a speed of 260 osc/min with horizontal stroke of 38 mm at room temperature. After shaking, the samples were centrifuged 15 min at 30 000 × g at 4°C. All supernatants were decanted into glass tubes, and diluted to 50 mL using 50 mM phosphate buffer. Homogenous mixing of sample extract and phosphate buffer was ensured by inverting the tubes 4–5 times.

SPE was used to remove matrix interference as much as possible from extracts. For the SPE, OASIS HLB Plus Short Cartridge and 20 port SPE vacuum manifold were used. A 20 mL reservoir was mounted above each cartridge to accommodate sample extract and chemicals for conditioning, washing, and elution. Sample extracts were loaded onto cartridges preconditioned with methanol, ultra pure water, and phosphate buffer, and vacuum was controlled to achieve a flow rate of 2–3 drops/s (Table 1). 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 4 min. Then pirlimycin was eluted sequentially with 3 mL methanol and 3 mL acetonitrile (Table 1), and the eluents were combined.

Eluted extracts were mixed by vortexing 30 s and 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 (N2) at 35°C using a Zipvap 20 evaporator. Dried extracts were completely dissolved in 1 mL methanol–water (50 + 50, v/v) with 0.1% formic acid by vortexing 30 s. Dissolved extracts were filtered through 0.2 µm PVDF syringe filter (Fisher) into 1.5 mL amber glass HPLC vials for the UPLC-MS/MS analysis.

UPLC-MS/MS Conditions and Pirlimycin Quantification

Pirlimycin was analyzed using Agilent 1290 UPLC coupled with Agilent 6490 Triple Quad tandem mass spectrometer. Electrospray negative ionization in multiple-reaction

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monitoring mode was used. Zorbax Extend C18 analytical column (4.6 × 50 mm, 5 µm particle size) coupled with Zorbax Extend C18 guard column (4.6 × 12 mm, 5 µm particle size) was used for chromatographic separation. Sampler and column compartments were kept at 8 and 40°C, respectively. The injection volume was 10 µL. A gradient elution program consisting of two mobile phases (mobile phase A, 0.1% formic acid in water; mobile phase B, 0.1% formic acid in methanol) were used at a flow rate of 0.5 mL/min to elute pirlimycin. Elution started with 30% mobile phase B, which then linearly increased to 95% mobile phase B at 6 min. The proportion of mobile phase B decreased linearly to 30% by 7.5 min and remained at 30% until the end of elution (12 min). MS parameters are listed in Table 2. Pirlimycin in positive and spiked samples were identified by comparing LC/MS/MS spectra of samples with those of pirlimycin standards. Accepted variation in mass to charge ratio and acceptable variation for the ratio of quantifier and qualifier ions was set at 20%. Seven different concentrations (0.5, 1, 2, 4, 5, 10, and 20 ng/mL matrix solution) of pirlimycin were used as external matrix-matched calibration standards, and the pirlimycin concentration in samples was quantified using the calibration curve of matrix-matched standards. Matrix-matched standards were prepared using eluted extract from blank feces or urine samples. Briefly, blank fecal or urine samples (containing no antibiotics) were extracted and cleaned up using the same method used to prepare matrix fortified and positive samples. Cleaned-up extract was spiked with diluted intermediate stock solution of pirlimycin to prepare a standard at a concentration of 100 ng/mL. This standard was then serially diluted with fecal or urine extracts to achieve different concentrations: 20, 10, 5,

4, 2, 1, and 0.5 ng/mL. One mL of each concentration was dried, redissolved, and filtered to achieve different concentrations of matrix-matched standards.

Method Validation

LOQ was determined using the equation:

LOQ = 10(SD/S)

where S is the slope of a calibration curve of seven matrix-matched standards (1, 2, 4, 5, 10, 20, and 50 ng/mL) and the SD is the standard deviation of responses from seven replicates of the lowest matrix-matched standard (1 ng/mL). A similar equation:

[LOD = 3.3(SD/S)]

was used to calculate LOD. Matrix effect was calculated using the equation:

[{(Pirlimycin in matrix/Pirlimycin in solvent)-1} × 100]%.

Instrument linearity was checked by analyzing 11 pirlimycin standards (1–750 ng/mL) prepared in methanol–acetonitrile (50 + 50, v/v). Triplicates of each concentration were injected three times. The calibration curve was constructed by plotting peak areas of the chromatogram against pirlimycin concentrations. Linearity was validated using calibration equation and correlation coefficients from regression analysis.

Spike recovery tests were performed by using matrix fortified samples. Matrix-fortified samples were prepared by spiking diluted intermediate stock solution of pirlimycin to 1 g feces (wet weight) or 1 mL urine before extraction, and in extracts (mid-spike). Three different spike concentrations were selected

Table 2. MS/MS operating conditions

Parameters

Ionization mode Electrospray negative ionization

Data collection Selected reaction monitoring

Nebulizer gas flow 16 L/min

Capillary voltage 3000 V

Fragmentation voltage 380 V

Collision energy 15 V

Ion source temperature 250°C

Precursor ion (m/z) 411

Qualifier ion (m/z) 363

Quantifer ion (m/z) 112

Table 1. SPE conditions

Step SolventVol., mL Destination

1 Conditioning Methanol 3 Discard

2 Conditioning Water 3 Discard

3 Conditioning Phosphate buffer (50 mM; pH 8.5) 3 Discard

4 Washinga Phosphate buffer (50 mM; pH 8.5) 2 Discard

5 Washinga Water 2 Discard

6 Elutiona Methanol 3 Collect

7 Elutiona Acetonitrile 3 Collecta Flow rate: 2–3 drops/s.

Table 3. Method validation data

Feces Urine

LOQ, ng/g or mLa 1.47 0.90

LOD, ng/g or ng/mLa 0.44 0.26

Pre-extraction spike recovery ± SD, %b

Spike level

2.5×LOQ 108 ± 3.1 98 ± 13

5×LOQ 93 ± 2.1 93 ± 5.7

10×LOQ 80 ± 1.8 89 ± 6.1

Post-extraction spike recovery ± SD, %b

Spike level

2.5×LOQ 106 ± 0.29 103 ± 2.9

5×LOQ 98 ± 4.2 89 ± 3.6

10×LOQ 94 ± 3.7 87 ± 8.8

Precision, %

Intra-day (n = 18)c 2.3–2.9 6.1–13

Inter-day (n = 36)d 2.3–5.6 7.8–14a ng/g wet feces or ng/mL urine.b SD = Standard deviation; n = 6.c Six replicates of three spike concentrations.d Three replicates of three spike concentration for 4 days.

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based on LOQ for pirlimycin in feces and urine matrix (22). For pre-extraction spike tests, 1 g feces or 1 mL urine was spiked with 1 mL of spike solutions (prepared in methanol) to achieve concentrations of 2.5, 5, and 10 LOQ. These were equilibrated 2 min before extractant was added to each spiked sample. The extraction, cleanup, and analysis procedures were as described in the previous section. For post-extraction recovery tests, fecal or urine extracts were spiked at concentrations of 2.5, 5, and 10 LOQ. Spiked extracts were equilibrated 2 min and vortexed

to achieve homogenous mixing followed by cleanup and analysis using procedures as described previously.

Intraday precision was evaluated by analyzing, at different times within a day, six replicates of pirlimycin-spiked blank samples (feces or urine) at three concentrations (2.5, 5, and 10 LOQ). Interday precision was assessed on four different days by preparing and analyzing three replicates of pirlimycin-spiked blank feces or urine at 4, 8, and 16 ng/kg for feces and 2.5, 5, and 10 ng/mL for urine. The matrix effect of feces and urine was evaluated by comparing peak response of seven pirlimycin standards dissolved in methanol–water (50 + 50, v/v, 0.1% formic acid) with those dissolved in blank fecal and urine extracts at a concentration range of 1–50 ng/mL.

All calibration standards were dried under N2 and redissolved in solvent using the same procedures as those used for samples to eliminate variation due to any loss of pirlimycin during the N2 drying process.

Results and Discussion

Optimization of Sample Extraction

Sample extraction and cleanup steps are closely associated because the approach to SPE is largely influenced by the type of extractant(s) used. But for simplicity, sample extraction optimization and the sample cleanup approach are discussed separately below.

To optimize sample extraction, blank water was spiked with pirlimycin. Acetonitrile was first used as the extractant because it was previously used to recover pirlimycin from bovine milk and tissue (18, 20). In this experiment, 50% acetonitrile in the presence of phosphate buffer (50 mM; pH 8.5) recovered 69% of pirlimycin from water. Similar recovery was observed when methanol was used at the same strength (70%) in the presence

Figure 1. (A) Blank fecal matrix-matched standard calibration

curve and solvent [methanol–water (50 + 50, v/v, 0.1% formic acid)]

standard calibration curve; (B) blank urine matrix-matched standard

calibration curve and solvent standard calibration curve.

Figure 2. Linearity of standard curve for pirlimycin standards

dissolved in methanol–water (50 + 50, v/v, 0.1% formic acid) at

concentrations ranging from 1 to 750 ng/mL.

Figure 3. UPLC-MS/MS SRM chromatograms for pirlimycin (A) in

a blank fecal sample (feces from a dairy cow not treated with

pirlimycin), (B) in blank feces spiked with pirlimycin at 10 LOQ,

(C) in a fecal sample collected from a dairy cow 4 h after pirlimycin

was administered, and (D) a pirlimycin standard (20 ng/mL)

dissolved in methanol–water (50 + 50, v/v, 0.1% formic acid).

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of phosphate buffer (50 mM; pH 8.5). During optimization of SPE cleanup, methanol appeared to be the better extractant and was thus selected for remaining experiments.

Next, pirlimycin extraction from feces was optimized to keep the solid to extractant ratio constant at 1:5 (w/v). When applied to the fecal matrix, 50% methanol with 50 mM phosphate buffer (pH 8.5) recovered <70% of pirlimycin spiked in 1 g wet feces. When methanol concentration was increased from 50 to 70%, almost complete pirlimycin recovery was achieved for both feces and urine.

Organic solvents at a strength as low as 10% were reported to be effective in eluting antibiotics from SPE cartridges (22). Therefore, prior to SPE, all extracts were diluted with phosphate buffer (50 mM) to 50 mL to bring final concentration of methanol in the extract below 10%.

Optimization of Sample Cleanup

SPE conditions were optimized using OASIS HLB cartridges because this cartridge was successfully used to reduce matrix effect during sample cleanup for analysis of pirlimycin in milk (18). To optimize SPE conditions, two extractants, acetonitrile and methanol, were tested on pirlimycin-spiked water. Different sequences and compositions of solvents were tested to optimize the SPE cleanup step. When acetonitrile extraction was used, conditioning of cartridges was optimized by testing sequential conditioning with acetonitrile followed by water or acetonitrile–formic acid (1 + 11, v/v, or 1 + 19, v/v) while washing (water and 3% acetonitrile) and elution (acetonitrile) solvents were kept fixed. Recovery was still <70%, but improved to 78% when 0.1% formic acid in water was used to wash SPE cartridges preconditioned with acetonitrile and acetonitrile–formic acid (1 + 19, v/v). Recovery was further improved (86%) and highest of all acetonitrile extraction approaches when pirlimycin was eluted from SPE cartridges using acetonitrile and methanol sequentially.

Pirlimycin in methanol extracts was diluted 10X with phosphate buffer (50 mM) and conditioning and washing of SPE cartridges involved phosphate buffer (50 mM). The highest recovery (>96%) was achieved when pirlimycin-spiked water was loaded onto SPE cartridges preconditioned sequentially with 2 mL methanol, 2 mL water, and 2 mL phosphate buffer. The cartridges were washed with 3 mL phosphate buffer and

3 mL water before pirlimycin was eluted from the cartridges using 3 mL methanol and 3 mL acetonitrile sequentially (Table 1). When this SPE cleanup approach was applied during analysis of pirlimycin-spiked blank fecal or urine samples, almost complete recoveries resulted (80–108% and 89–98% for feces and urine, respectively; Table 3).

Method Qualitation and Validation

Calibration curves prepared from calibration standards dissolved in LC mobile phase had higher slope values as compared to curves of pirlimycin dissolved in feces or urine. This finding indicated a matrix effect for both feces and urine (Figure 1). Both fecal and urine matrix caused ion suppression, and the matrix effect was larger for urine than for feces (–78 vs –9%). Calibration standards were prepared on the day of analysis. LOQ for pirlimycin in pirlimycin-spiked blank bovine feces and urine was 1.47 ng/g (wet weight) and 0.90 ng/mL, respectively.

To the best of our knowledge, this report is the first ever published on LOQ for UPLC-MS/MS quantification of pirlimycin in bovine feces and urine. In this experiment, matrix-matched calibration standards were used to reduce any bias on pirlimycin quantification by accounting for background matrix of feces and urine (Figure 1A and B). The instrument response was linearly correlated (r2 = 0.9992) with pirlimycin concentration within the range from 1 to 750 ng/mL (Figure 2). All the standard curves used for pirlimycin quantification in samples were within this range (Figure 1). Figures 3 and 4 show the chromatograms of blank feces or urine, pirlimycin spiked in blank feces or urine, feces and urine collected 4 h following pirlimycin administration, and pirlimycin in solvent.

The accuracy of the method was evaluated by testing recovery of pirlimycin spiked in blank feces and urine (collected from cows not treated with pirlimycin). Spike recovery tests were performed either by spiking pirlimycin in feces and urine (pre-extraction) or by spiking in sample extract (post-extraction). The efficiency of the entire method, including extraction, cleanup, and quantification steps, was assessed by using pre-extraction (pirlimycin spiked in blank feces or urine before extraction) recovery tests. Post-extraction (pirlimycin spiked in the extracts of blank feces or urine) recovery tests were used to evaluate the efficiency of the steps from SPE to

Figure 4. UPLC-MS/MS SRM chromatograms for pirlimycin (A) in a blank urine sample (urine from a dairy cow not treated with pirlimycin),

(B) in blank urine spiked with pirlimycin at 10 LOQ, and (C) in a urine sample collected from a dairy cow 4 h after pirlimycin was administered.

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quantification. Pre-extraction recovery of pirlimycin in blank feces and urine samples ranged from 80 to 108% and from 89 to 98%, respectively (Table 3). Post-extraction recovery of pirlimycin spiked in the extracts of blank feces and urine ranged from 94 to 106% and from 87 to 103%, respectively (Table 3). The recoveries for both pre- and post-extraction spike recovery tests indicated high efficiency of extraction and high accuracy of the method.

The precision of the method was evaluated in terms of repeatability (intraday variation, % RSD), and reproducibility (interday variation, % RSD). Repeatability of the method was from 2.3 to 2.9% and from 6.1 to 13% for feces and urine, respectively (Table 3), and was within the acceptable range of method repeatability set by the U.S. Food and Drug Administration (23). The reproducibility values ranged from 2.3 to 5.6%, and from 7.8 to 14% for spiked blank feces and urine, respectively (Table 3).

Applicability of the Method

Applicability of the developed analytical approach was evaluated by analyzing hourly fecal and urine samples collected at 4, 6, and 10 h, and daily samples collected from total daily fecal and urine output for 5 days from dairy cows following intramammary infusion (50 mg/cow) of a pirlimycin-based

antibiotic (PIRSUE; Zoetis, Florham Park, NJ). With the application of this method, we were able to detect pirlimycin at a range of 64.7–287 ng/g (wet weight) in the feces collected (Table 4). Pirlimycin was detected in urine samples, and urinary concentrations of pirlimycin ranged from 46.1 to 254 ng/mL (Table 4). Reported pirlimycin concentrations in feces and urine were not normalized with their respective recoveries shown in Table 3.

Conclusions

An UPLC-MS/MS-based qualitative and quantitative method for pirlimycin in bovine feces and urine was developed and validated. Optimized sample extraction and SPE cleanup allowed qualitative and quantitative detection of pirlimycin in bovine feces and urine with very low LOQ. This method can be applied to qualification and quantification of trace amounts of pirlimycin in bovine feces and urine with high accuracy. This method will be an important contribution to environmental or pharmacokinetics studies where quantification of pirlimycin in bovine feces and urine at nanogram or subnanogram concentration is required.

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Table 4. Quantification of pirlimycin in feces and urine collected from dairy cows following intramammary infusion

of pirlimycin

Hours post-treatmenta, h Concentration

(ng/g or mL ± SDb)

Hourly samplesc

Feces 1 4 65 ± 1

Feces 2 6 61 ± 4

Feces 3 10 72 ± 2

Urine 1 4 231 ± 2

Urine 2 6 254 ± 4

Urine 3 10 153 ± 7

Daily samplesd

Feces 4 24 152 ± 1

Feces 5 48 287 ± 3

Feces 6 72 174 ± 8

Feces 7 96 84 ± 1

Feces 8 120 41 ± 6

Urine 4 24 232 ± 5

Urine 5 48 198 ± 12

Urine 6 72 113 ± 3

Urine 7 96 72 ± 2

Urine 8 120 46 ± 1a Hours after dairy cows were treated with first dose of pirlimycin.b n = 3.c Hourly samples are spot sampling at 4, 6, and 10 h post-treatment.d Daily samples are sub-samples collected from total daily fecal and

urine output.

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