Seminar2019 Paper 6 in Cell Solid Liquid Extraction.Pdf

Home , Moroxydine

Journal of Chromatography A, 1488 (2017) 10–16

Contents lists available at ScienceDirect

Journal of Chromatography A

jo urnal homepage: www.elsevier.com/locate/chroma

Development and application of an in-cell cleanup pressurized liquid

extraction with ultra-high-performance liquid

chromatography-tandem mass spectrometry to detect prohibited

antiviral agents sensitively in livestock and poultry feces

a b b c b a

Huizhen Wu , Jianmei Wang , Hua Yang , Guoqin Li , Yinhuan Zeng , Wei Xia ,

a,∗ b,∗

Zuguang Li , Mingrong Qian

College of Chemical Engineering, Zhejiang University of Technology, Hangzhou, 310014, China

Institute of Quality and Standard for Agro-products, Zhejiang Academy of Agricultural Sciences, MOA Key Laboratory for Pesticide Residue Detection,

Hangzhou, 310021, China

Institute of Animal Husbandry and Veterinary Science, Zhejiang Academy of Agricultural Science, Hangzhou 310021, China

a r a

t b

i c l e i n f o s t r a c t

Article history: An in-cell cleanup pressurized liquid extraction was developed to analyze prohibited antiviral agents in

Received 12 September 2016

livestock and poultry feces. Extraction and cleanup were integrated into one step. The extraction was per-

Received in revised form ◦

formed using methanol-acetonitrile (1:1, v/v) with 0.5% glacial acetic acid at 90 C, and 0.75 g of PSA was

24 December 2016

used as the adsorbent during the extraction procedure. Under optimal conditions, the average recoveries

Accepted 26 January 2017 −1

for 11 antiviral drugs were 71.5–112.5% at three spiked levels (20, 40, and 100 ␮g kg ). The detection

Available online 27 January 2017

limits and detection quantitations of the analysis method for the eleven antiviral drugs were 0.6–1.4 and

−1

1.4–4.7 ␮g kg , respectively. Finally, the method was applied to analyze amantadine, oseltamivir and

Keywords:

its metabolites oseltamivir acid in duck feces based on an experiment of an oral dose of two antiviral

Antiviral agents

drugs in duck. The amantadine, oseltamivir and oseltamivir acid can be detected in feces within approx-

Pressurized liquid extraction

Livestock and poultry feces imately four weeks after amantadine and oseltamivir were orally administered. The results indicate that

the residue analysis in feces is a noninvasive method to monitor inhibited antiviral agents efﬁciently in

livestock and poultry breeding.

1. Introduction object to monitor the antiviral-drug usage during the process of

livestock and poultry breeding. Therefore, a sensitive method to

Antiviral agents such as amantadine, rimantadine, moroxydine, analyze the antiviral agents in livestock and poultry feces must be

oseltamivir and acyclovir have been banned in poultry farming established.

in China to prevent interference with the preventive effect of Several analyte methods have been reported for the determi-

vaccinum. Nevertheless, these antiviral agents remain illegally nation of the residues of these compounds in solid samples, most

occasionally used for the pretreatment of avian inﬂuenza in poul- of which focus on animal-derived food [1,3,9–13]. Solid-phase

try farming in China [1,2]. Low levels of amantadine have been extraction [1,3,11,13] and QuEChERS method [9,10] have been

detected in poultry muscle and liver [1,3,4]. Some measures should reported for sample preparation. The pressurized liquid extraction

be proposed to monitor these residues in the livestock and poul- (PLE) method is a simple, rapid and promising sample preparation

try cultivation process to guarantee the safety and quality of animal method and has been widely applied for multi-residue determi-

foods. It is reported that after the administration of antiviral agents, nation in complex matrices including animal and environment

some of them are excreted in non-metabolized form [5] or as active samples. Hussen et al. [14] have packed adsorbents in the extrac-

metabolites in feces [6–8]. Thus, animal feces can be selected as an tion cells to combine the extraction and puriﬁcation into one step

for the analysis of organochlorine pesticides in soil. This method

has been efﬁciently applied to determine multi-pesticides [14–19],

persistent and emerging organic pollutants [17,18,20–28]. Never-

∗

Corresponding authors. theless, only a few studies focused on the possibility of extending

E-mail addresses: [email protected] (Z. Li), [email protected] (M. Qian).

http://dx.doi.org/10.1016/j.chroma.2017.01.070

H. Wu et al. / J. Chromatogr. A 1488 (2017) 10–16 11

this approach to residue analysis in feces [29]. As far as we know, To separate the antiviral agents, gradient elution was performed

this method has not been applied for antiviral residues detec- on a BEH-HILIC HPLC column (2.1 × 100 mm, 1.7 ␮m, Waters) at

◦ −1

tion in agricultural foods or environment samples. This work is to 30 C. The mobile phases were acetonitrile (A) and 5 mmol L

develop a simple and sensitive method to analyze antiviral agents ammonium acetate water with 0.1% formic acid (B). The starting

in livestock and poultry feces using ultra-high-performance liq- mobile-phase composition was 5% B. After an isocratic step at 5%

uid chromatography-tandem mass spectrometry (UPLC–MS/MS) to B for 2 min, B was linearly increased to 45% in 7 min, decreased

trace the misuse. Then, the regulatory authorities can take appro- to 5% in 3 min, and held constant for 3 min; the total cycle time

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priate actions to prevent the abuse of antiviral drugs. was 13 min. The mobile-phase ﬂow rate was 0.2 mL min , and the

injection volume was 2 ␮L.

2. Materials and methods For the mass spectrometer, the positive electrospray ioniza-

tion mode and multiple-reaction monitoring (MRM) mode were

2.1. Reagents and materials used. The optimal conditions for the sample analysis were as fol-

lows: ion-spray voltage: 5000 V; curtain gas: 40 psi (nitrogen); ion

◦

HPLC gradient grade acetonitrile and methanol were purchased source temperature: 500 C; ion source gas 1 and ion source gas 2:

from Merck (Germany)and HPLC grade glacial acetic acid was pur- 50 psi. The MRM transition parameters for the quantiﬁcation are

chased from Scharlau (Spain). The sorbents were purchased from presented in Table 1.

Agela Technologies (China): C18 (40 ␮m particle size), primary sec-

ondary amine (PSA) (40–60 ␮m particle size), silica gel (70–90 ␮m 2.4. Quantiﬁcation and method validation

particle size) and Florisil (60–100 mesh). Diatomaceous earth with

80–100 mesh was purchased from Thermo Scientiﬁc (USA). Neutral Concentrations of the amantadine, rimantadine, memantine,

alumina was from Aladdin Industrial (China). Puriﬁed water with somantadine, acyclovir, imiquimod and ganciclovir in the sam-

resistivity higher than 18.2 M cm was obtained by a Milli-Q water ples were performed using internal standard method, which were

puriﬁcation system (Millipore, USA). added to the sample post extraction. The calibration curves (three

The standards of amantadine and oseltamivir acid were replicates) were using the levels 0.5, 1.0, 2.0, 5.0, 10, 20, 50

−1

␮

purchased from Anpel Laboratory Technologies (Shanghai) Inc and 100 g L with a constant internal standard concentration

−1

␮

(China). Rimantadine, memantine, oseltamivir phosphate, acy- of 20 g L . For oseltamivir, oseltamivir acid, moroxydine and

clovir, imiquimod, ganciclovir, arbidol and the internal standards, arbidol, the concentrations were determined using the matrix

amantadine-d15, rimantadine-d4, memantine-d6 and acyclovir- match calibration method. The linear portion of the standard curve

d4 were purchased from Toronto Research Chemicals (Canada). was used to quantify antiviral agents in the extracts; if the concen-

Moroxydine was purchased from Sigma (USA). Somantadine was trations were above the linear portion, a higher internal standard

purchased from J & K Scientiﬁc (USA). was added to the samples, and the extracts were subsequently

The stock solutions were prepared as individual diluted and analyzed.

−1

100–200 ␮g mL solutions in acetonitrile or acetonitrile aqueous All generated data from the analysis were subject to strict qual-

◦

solution. They were stored in 4 C without light and found to ity assurance and quality control (QA/QC) procedures. With each

be stable for at least two month. Work standard solutions were batch of six samples to be analyzed, a solvent blank, a procedure

freshly prepared by diluting the stock solutions with acetonitrile. blank and a spiked sample were sequentially run to ensure accurate

Corrections were made for salt (HCl or H3PO4 salts) and purity. and precise measurement. A blank injection was performed after 3

sample injections to ensure that there was no carry-over. A check

2.2. In-cell cleanup pressurized liquid extraction procedure standard injection was performed after 20 injections to conﬁrm the

instrument performance, and the computed concentration must be

The antiviral agents from livestock and poultry feces were within 20% of the expected value.

extracted by PLE using an ASE 350 system (Thermo Scientiﬁc, USA). The limit of detection (LOD) and limit of quantitation (LOQ) for

The sample puriﬁcation was simultaneously performed with the the method were estimated from the data of the recovery test with

extraction (in-cell cleanup) by placing the sorbent inside the cell the lowest spiked concentration where an S/N could be 3 and 10,

(PSA). At the bottom of the 34-mL stainless steel vessel was a cellu- respectively.

lose ﬁlter; from down to up, there were 2 g of diatomaceous earth, The recovery studies were carried out in blank samples spiked

0.75 g of PSA, 0.5 g of freeze-dried sample and 1 g of diatomaceous with a suitable volume of working standard solution. Then they

earth. Subsequently, the cells were tightly closed and inserted into were allowed to stand at room temperature for 1 h and main-

◦

the cell tray for the extraction and cleanup. The optimal ASE con- tained at 4 C for 24 h. The matrix effects were calculated by spiking

−1

␮

ditions were as follows: extraction solvent: methanol-acetonitrile the standard solutions at a concentration of 50 g L for each

(1:1, v/v) with 0.5% glacial acetic acid; extraction temperature: compound into the blank sample extracts and comparing to the

◦

90 C; heating time: 5 min; extraction pressure: 1500 psi; static concentration in the solvent. The values above or below 100% indi-

extraction period: 15 min; static extraction cycles: one, rinse vol- cate signal enhancement or suppression, respectively.

ume, 30%; pressurized nitrogen purge time: 120 s. The sample The precision was expressed as the relative standard deviation

extract was condensed to dryness using a Turbovap LV (Biotage, (RSD). The intra-day and inter-day RSDs were examined at three

Sweden) with a nitrogen stream. The residue was dissolved in 1 mL concentration levels. The run-to-run repeatability was analyzed on

initial mobile phase and transferred into a 1.5 mL tube. The tube the same day, and the day-to-day reproducibility was analyzed on

was centrifuged at 10000 rpm for 3 min, and the supernatant was six consecutive days.

collected and ﬁlter through a 0.22 ␮m ﬁlter for analysis.

2.5. Real sample collection

2.3. Instrumentation and chromatographic conditions

Fifteen Shaoxing ducks (1.41 ± 0.09 kg), a popular variety with

The LC system consisted of an LC 30AD ultra-performance liquid high egg yields in China, were purchased from the local market.

chromatography from Shimadzu (Japan), which was coupled with The ducks were randomly divided into three groups and raised in

a 6500 QTRAP mass spectrometer (Applied Biosystems, USA) and stainless-steel crates. The control feces were collected 24 h prior to

equipped with the Multiquant V.3.0 data-processing software. dosing. After the 24-h acclimation period, those ducks were admin-

12 H. Wu et al. / J. Chromatogr. A 1488 (2017) 10–16

Table 1

Target compounds and their optimized LC–MS/MS parameters on positive ionization mode, and the internal standards assigned for their quantiﬁcation.

Compound Precursor ion (m/z) Product ion (m/z) Declustering potential (v) Collision energy (v) Corresponding internal standard

Amantadine 152 135 60 23 Amantadine-d15

93 60 36 a

Rimantadine 180.4 121 30 31 Rimantadine-d4

163 30 20 a

Memantine 180.4 107 30 34 Memantine-d6

163 30 20 a

Somantadine 208 135 40 27 Rimantadine-d4

191 40 13 a

Oseltamivir 313 225 40 19

166 40 14

Oseltamivir acid 285.2 197 50 12

137.9 50 25 a

Acyclovir 226 152 30 38 Acyclovir-d4

135 30 16 a

Imiquimod 241 185 50 33 Rimantadine-d4

168 50 44 a

Moroxydine 172 113 40 27

130 40 23 a

Ganciclovir 256 152 40 17 Acyclovir-d4

135 40 41 a

Arbidol 477 432 60 25

279 60 46

Amantadine-d15 167 150 60 27

Memantine-d6 186 169 30 16

Rimantadine-d4 184 167 30 23

Acyclovir-d4 230 152 30 16

The transition ion pair used for quantitation.

istered an oral dose of amantadine and oseltamivir in a gelatin umn, better chromatographic separations for oseltamivir acid and

−1 −1

capsule at approximately 4 mg kg body weight and 3.7 mg kg interferences in the spiked sample were obtained when the HILIC

body weight for ﬁve days, respectively. Throughout the study column was used (Fig. 1). Therefore, to well separate the targets

period, feed and water were available ad libitum. The feces were and guarantee accurate quantitation of the interferences, the HILIC

collected in stainless-steel trays at the bottom of the crates. All feces column was selected to detect the target compounds in further

in the stainless-steel crates were thoroughly mixed and collected study, and the chromatogram of eleven antiviral agents with good

every 24 h. The collected feces were freeze-dried and ground to a separation effects is shown in Fig. 2.

ﬁne powder. The animal experiments were complied with the pol-

icy on the care and use of laboratory animals and approved by the

Institutional Animal Care and Use Committees, Zhejiang Academy

3.2. Optimization of the in-cell cleanup PLE method

of Agricultural Sciences, China.

3.2.1. Optimization of the sorbent and extraction solvent

3. Results and discussion To extract the maximum target analyte with minimum interfer-

ences, three solvents and ﬁve different sorbents were tested using

3.1. Optimization of ultra-high-performance liquid a full factorial design. Various adsorbent materials were tested to

chromatography perform the in-cell cleanup while eluting using methanol, acetoni-

trile or methanol-acetonitrile (1:1, v/v). Glacial acetic acid has been

For practical reasons, acyclovir, moroxydine and ganciclovir are added in solvent for antiviral drugs extract in chicken tissues [4,11]

hydrophilic compounds and difﬁcult to retain in conventional HPLC and efﬁcient recoveries were obtained. With this reference, extrac-

columns [1,11]. Then, to better separate the target compounds, we tion solvent containing 0.5% glacial acetic acid was tried in this PLE

investigated four different HPLC columns. In this part, four dif- procedure. The tested sorbents were alumina, silica, PSA, C18 and

ferent types of LC columns were tested: BEH C18 HPLC column Florisil. The cleanup materials and elute solvent were tested in the

−1

␮

(2.1 × 100 mm, 1.7 ␮m), BEH-Amide HPLC column (2.1 × 100 mm, recovery experiment with a spiking level of 40 g kg in the blank

␮

1.7 m), HSS-T3 HPLC column (2.1 × 150 mm, 1.8 ␮m) and BEH- feces. The spiked samples were exposed to the target compounds

◦

HILIC HPLC column (2.1 × 100 mm, 1.7 ␮m). In the positive mode, for 24 h prior to extraction. The extraction was performed at 60 C,

mobile phase A was ﬁxed to acetonitrile, and mobile phase B was 1500 psi and 3 extraction cycles (5 min per cycle). When Florisil was

−1

ﬁxed to 5 mmol L ammonium acetate water with 0.1% formic used, the recoveries of the target compound were almost the lowest

acid. Using the C18 and T3 columns, acyclovir, moroxydine and gan- possibly because the Florisil in the organic extracts preferentially

ciclovir were eluted at the dead time of the system and showed retained polar antiviral agents in additional to lipid interferences

widened and tailed chromatographic peaks. Instead, good chro- [15]. Compared with methanol and acetonitrile, the methanol-

matographic retentions of those three compounds were obtained acetonitrile (1:1, v/v) gave almost the best extract efﬁciency when

from both Amide column and HILIC column (Fig. 1). For acyclovir alumina, silica, PSA and C18 were used (Fig. S1). As shown in Fig. 3,

and ganciclovir, the responses on the HILIC column were better than when methanol-acetonitrile (1:1, v/v) was used, the PSA and C18

those on the Amide column; meanwhile, the response of moroxy- gave similar recovery values for all targets except arbidol. With PSA,

dine on the HILIC column was slightly below that on the Amide higher recovery for arbidol was obtained. With the results from this

column. In addition, when the feces samples were analyzed for the optimization step, methanol-acetonitrile (1:1, v/v) with 0.5% glacial

recovery studies on the Amide column, a widened chromatographic acetic acid and PSA were selected for further optimization of the PLE

peak appeared for arbidol (Fig. 1B). Compared with the Amide col- method.

H. Wu et al. / J. Chromatogr. A 1488 (2017) 10–16 13

−1.

Fig. 1. Comparison between BEH-HILIC (A) and BEH-Amide (B) chromatogram columns in blank feces sample spiked at 20 ␮g kg

3.2.2. Optimization of the PLE method

|Recovery − |

This PLE method involves selecting the extraction tempera- Thus, we used the equation: S = %i 100 to calculate

ture, extraction time, number of cycles and amount of sorbent. i=1

To identify the optimum PLE factors to improve the experimen- the output variable; a smaller value of S represents better experi-

13 13

tal performance, an L27 (3 ) orthogonal array was used in this ment performance. The obtained experimental data from L27 (3 )

◦

study. Four factors at three levels were studied: temperature (60 C, optimization (shown in the Support information Table S1) were

◦ ◦

90 C, and 120 C), extraction time (5 min, 10 min, and 15 min), cycle evaluated using the ANOVA statistical method and range analysis

index (1–3) and PSA amount (0.25 g, 0.5 g, and 0.75 g). For each (R values in Table S1). A larger R value corresponds to a stronger

experimental trial, two replicate experiments were performed. effect of the test factor on the result. Table S1 shows the follow-

Thus, negative animal feces spiked with the target compounds at ing factors sorted by the effect on the result in a descending order:

−1

50 ␮g kg were prepared and analyzed by the described method the PSA amount (D), interaction between extraction temperature

according to the L27 (3 ) orthogonal array, in replicate. The total and extraction time (A B), extraction temperature (A), extraction

amount of spiked samples was 54. Then the PLE was conducted time (B) and cycle index (C). First, the effect of the PSA amount

with 1500 psi extraction pressure, 30% rinse volume and 120 s of was the most important, which the ANOVA results (Table S2) also

nitrogen purge time. The recoveries of the target compounds were indicate (P < 0.05). The best PSA amount was identiﬁed in the level

calculated based on the matrix calibration curves under different analysis. Compared with K1 and K2 in column 9 in Table S1, K3

conditions. A synthetically output variable was required to evaluate achieved the lowest sum of S. Therefore, the selected amount of

the effect of the associated level of each factor in the experiment. PSA was 0.75 g. Second, the R value in column 5 in Table S1 was

14 H. Wu et al. / J. Chromatogr. A 1488 (2017) 10–16

−1.

Fig. 2. UPLC-MRM chromatograms of the investigated antiviral agents in a feces sample spiked at 20 ␮g kg

Fig. 3. Extraction recoveries for selected target compounds using different sorbents in methanol-acetonitrile (1:1, v/v). The error bars represent the standard deviation (n = 3).

◦

larger than that in column 1, which indicates that the interaction 90 C for the extraction temperature, 15 min for the static extrac-

of the extraction time and extraction temperature should be con- tion period, one static extraction cycle and 0.75 g of sorbent. The

sidered with priority compared to the extraction temperature. The average S was 169, which is lower than the other test results in

◦

ANOVA results (Table S2) also indicate that the effect of the inter- Table S1. Thus, a 90 C static extraction for 15 min in 1 cycle with

action between extraction temperature and extraction time was 0.75 g PSA was selected in further studies.

signiﬁcant (P < 0.05) to the output variable. Lower S was achieved

◦

at a middle temperature (90 C) in a relatively longer extraction

◦ 3.3. Method validation

time (15 min). Therefore, 90 C was selected as the extraction tem-

perature; for the compromise level, 15 min was selected as the

The performance of the method was evaluated under the

extraction time. Because the R value for the cycle index is notably

optimized conditions including the linearity, matrix effect, pre-

small compared to the other factors, it was concluded that the

cision, LOD and LOQ for all analyzed compounds. For the matrix

effect of this factor is small and can be neglected in this study. To

effect, serious signal suppression was observed for somantadine

save time, the cycle index was selected as 1 time. Three parallel

and moroxydine with values more than 10%. Thus, the stable

experiments were performed under the condition of A2B3C1D3, i.e.,

isotopic internal standards were used to correct for the losses

H. Wu et al. / J. Chromatogr. A 1488 (2017) 10–16 15

Table 2

Analytical method validation parameters: recoveries for target compounds, the limit of detection (LOD) and the limit of quantiﬁcation (LOQ).

−1 −1

␮ ␮

Compound %Recoveries (% RSD) (n = 5) LOD ( g kg )LOQ( g kg )

−1 −1 −1

20 ␮g kg 40 ␮g kg 100 ␮g kg

Amantadine 88.2(7.9) 86.1(7.7) 88.7(2.6) 0.6 2.0

Rimantadine 91.9(8.9) 111.0(6.7) 98.4(4.3) 0.7 2.4

Memantine 98.6(12.7) 96.5(9.9) 89.3(5.8) 0.7 2.4

Somantadine 85.3(12.9) 76.4(8.2) 95.6(3.8) 0.8 2.5

Oseltamivir 78.7(5.8) 87.2(3.9) 92.7(2.4) 0.9 3.0

Oseltamivir acid 82.3(8.4) 73.6(9.1) 90.3(7.5) 1.4 4.7

Acyclovir 97.1(11.7) 85.2(9.6) 98.6(4.3) 0.5 1.8

Imiquimod 112.5(5.7) 98.2(3.0) 97.1(3.6) 0.4 1.4

Moroxydine 94.2(8.3) 96.9(4.5) 95.4(5.9) 1.2 4.1

Ganciclovir 71.5(9.6) 85.5(8.4) 90.3(5.8) 0.7 2.2

Arbidol 75.4(11.6) 68.3(7.8) 89.5(9.0) 1.2 3.9

−1

Fig. 4. Fecal concentration versus time proﬁles for amantadine, oseltamivir and oseltamivir acid (␮g kg dry weight). The error bars represent the standard deviation of the

three groups.

and matrix effect. In this study, amantadine-d15, rimantadine- these banned agents that were applied during livestock and poul-

d4, memantine-d6 and acyclovir-d4 were obtained; according to try breeding. The variety trend of the oseltamivir acid residue, as

the chromatographic retention time and structure, they were the active metabolite of oseltamivir, in the duck feces was similar to

applied to minimize the matrix effect to quantitate amantadine, that of its parent. For the ﬁrst seven days, the residue concentrations

rimantadine, somantadine, imiquimod, memantine, acyclovir and in feces were high, and the extracts were diluted and determined

ganciclovir residues in feces (Table 1), whereas the matrix match using the matrix match calibration method. The concentrations of

standards were applied to compensate the matrix effects to quan- amantadine, oseltamivir and oseltamivir acid in feces increased

titate oseltamivir, oseltamivir acid, moroxydine and arbidol. Good during the ﬁrst 2 days, decreased on day 3, peaked at day 4 and

2 −1

linearity was obtained with R > 0.99 for both internal standard began to decrease to below 30 ␮g kg on day 13. After 14 days, the

method and matrix match calibration method for all compounds three residues concentrations were maintained at low levels for

−1

␮

over the concentration range of 1–100 g L . As shown in Table 2, another two weeks (Fig. 4). Over approximately four weeks after

the average recoveries for antiviral agents were 71.5–112.5% at the oral dose of amantadine or oseltamivir, the average concentra-

−1 −1

␮

three spiked levels (20, 40, 100 g kg ), where the intra-day RSD tions of amantadine in the feces ranged from 5.4 ± 0.9 ␮g kg to

−1

(n = 6) was 2.4–12.9%, and inter-day RSDs were 5.7–14.1%, which 15763 1478 ␮g kg . The oseltamivir concentration ranged from

−1 −1

indicates the good accuracy and precision of the developed method. 12431 ± 1932 ␮g kg to 4.2 ± 0.4 ␮g kg , and oseltamivir acid

−1 −1

For the test compounds that were determined at a fortiﬁcation level ranged from 24095 ± 4386 ␮g kg to 11.8 ± 1.3 ␮g kg .

−1 −1

of 20 ␮g kg , the LODs and LOQs were 0.6–1.4 and 1.4–4.7 ␮g kg , respectively.

4. Conclusions

3.4. Application to real samples A procedure of one-step extraction and cleanup sample prepara-

tion with UPLC–MS/MS to determine 10 prohibited antiviral agents

The optimized in-cell cleanup PLE UPLC–MS/MS procedure was and one metabolite in livestock and poultry feces was successfully

applied to the collected feces samples from the amantadine- and developed and applied to analyze those prohibited antiviral agents

oseltamivir-administered ducks. The objective of this treatment in the manure-based organic fertilizer. This method was also used

was to assess the applicability of the developed method to monitor to analyze amantadine, oseltamivir and its metabolites oseltamivir

16 H. Wu et al. / J. Chromatogr. A 1488 (2017) 10–16

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