Analytical and Bioanalytical Chemistry

, Volume 410, Issue 22, pp 5555–5565 | Cite as

Simultaneous determination of amantadine and rimantadine in feed by liquid chromatography-Qtrap mass spectrometry with information-dependent acquisition

  • Qi Jia
  • Dan Li
  • Xinlu Wang
  • Shuming Yang
  • Yongzhong Qian
  • Jing Qiu
Research Paper
Part of the following topical collections:
  1. Food Safety Analysis


A sensitive method for simultaneous determination of amantadine and rimantadine in feed was developed using an ultra-high-performance liquid chromatography-triple quadrupole linear ion trap mass spectrometry (UHPLC-Qtrap-MS) in the multiple reaction monitoring information-dependent acquisition-enhanced product ion (MRM-IDA-EPI) mode, and employing the mixed cation exchange (MCX) solid-phase extraction column as sample cleanup and amantadine-d15 and rimantadine-d4 as internal standards, respectively. Compared to traditional MRM mode, for the targeted drugs in feed simultaneously both the secondary mass spectra and MRM information can be obtained using UHPLC-Qtrap-MS with MRM-IDA-EPI mode, and thus more accurate qualitative confirmation results achieved even at lower concentration of 0.2 μg/L in acceptable purity fit values. After optimization of sample preparation, good linearities (R > 0.9994) were obtained over the concentration range from 1 to 200 μg/L for amantadine and rimantadine. The precision was validated by intra-day and inter-day, and the relative standard deviations were all within 9.61%. Mean recoveries ranged from 76.1 to 112% at spiked concentrations of 0.5–100 μg/kg in three types of feed samples, including formula feed and complex concentrated feed for pigs and premix feed for chicken. The limits of detection (LODs) and quantification (LOQs) were 0.2 and 0.5 μg/kg for both drugs, respectively. The application in real feed samples further proved the accuracy and reliability of the developed method. This method provides an important tool to detect illegal uses of amantadine and rimantadine in feed.

Graphical abstract

Simultaneous quantitation and qualitative confirmation of amantadine and rimantadine in feed by MRM-IDA-EPI


Amantadine Rimantadine Feed Solid-phase extraction Liquid chromatography-triple Qtrap mass spectrometry 


Antiviral drugs including amantadine (1-adamantanamine) and rimantadine (1-(1-adamantyl)ethanamine) have been widely used for clinical prevention and treatment of viral infections for animals especially by the influenza A virus [1]. In 2012, “fast-growing chicken” was reported by Chinese media because of using antiviral drugs in the process of breeding to protect flocks from influenza infection [2]. The illegal use of antiviral drugs can lead to their residue in animal-derived products [3], and further lead to immunosuppression toxicity for animals and potential drug resistance for human beings [4]. This can bring adverse consequences in prevention and control of major animal diseases, and therefore is of higher public health concern. Thus, many countries including USA [5] and China [6] set strict rules to prohibit the use of these drugs from livestock and poultry farming. Feed is the most important source of food for farmed animals, and some drugs and additives were often added in feed for breed animals for clinical treatment and as nutrient supplement. The feed can be one of the most likely sources which introduce antiviral drugs into the animal body. Until now, there is no analytical method for determine of amantadine and rimantadine in feed. Therefore, it is necessary to develop and validate an effective method for the analysis of these drugs in feed and help with preventing their illegal use.

Some reports involved the determination of antiviral drugs in different matrices except for feed covers biological fluids [7, 8, 9, 10, 11], animal tissues [12, 13, 14], and eggs [15, 16]. Several instruments were used to detection such as capillary zone electrophoresis [17], gas chromatography-mass spectrometry (GC-MS) [8], high-performance liquid chromatography with ultraviolet detection (HPLC-UV) [18], fluorescence detection (HPLC-Flu) [19], high-performance liquid chromatography-triple quadrupole mass spectrometry (LC-MS/MS), and ultra-high-performance liquid chromatography-triple quadrupole mass spectrometry (UHPLC-MS/MS) [12, 13, 15]. Thereinto, LC-MS/MS and UHPLC-MS/MS exhibited the excellent advantages of higher selectivity and better sensitivity, and simple sample preparation and direct determination of amantadine [16, 20] and rimantadine [16, 21] without derivatization. With these methods, some positive samples for amantadine were found with concentrations of 1.79–86.0 μg/kg in chicken and eggs [12, 13, 14, 15]. Additionally, ultra-high-performance liquid chromatography-triple quadrupole linear ion trap mass spectrometry (UHPLC-Qtrap-MS) can obtain quantitative multiple reaction monitoring (MRM) information and qualitative QTRAP scans in one run [22]. Compared to traditional triple quadrupole mass spectrometry that only can obtain quantitative MRMs, UHPLC-Qtrap-MS can obviously get more mass spectrum information for more accurate confirmation of targeted analytes.

Sample preparation methods play an important role in the determination of antiviral drugs because fast and effective extraction and cleanup of different types of matrices are needed. One of the classic methods for determination of veterinary drugs in food is liquid-liquid extraction combined with solid-phase extraction (SPE), which can obtain more purification effects and less interference but costs more time for sample preparation. In recent years, the QuEChERS (quick, easy, cheap, effective, rugged and safe) method was regarded as a simple and fast sample pretreatment method, and widely used for determination of veterinary drugs in different food matrices [12, 13, 14, 15, 23]. Our previous reports also successfully applied QuEChERS method to detect antiviral drugs including amantadine and rimantadine in animal tissues [14, 15] and eggs [14], and obtained good accuracy and sensitivity. What is challenging for feed that is composed with a variety of ingredients, and comparatively more complicated than other food matrices like tissues and eggs. However, the traditional QuEChERS method did not work well for determination of amantadine and rimantadine proved by our pre-experiment. The SPE method was often used to for analysis of veterinary drugs and additives in feed [24] and other complicated samples [2, 25], and showed better cleanup effects compared to QuEChERS method in general. The aim of this study is to develop and validate a new method for simultaneous quantitation and qualitative confirmation of amantadine and rimantadine in feed by UHPLC-Qtrap-MS. In consideration of complicated matrix and the polarity of amantadine and rimantadine, the mixed cation exchange (MCX) SPE columns were employed for sample cleanup [2, 25].

Materials and methods

Chemicals and reagents

The standards of amantadine hydrochloride (purity 98%) and rimantadine (purity 99%) were obtained from Alta Technology (Tianjin, China). Amantadine-d15 hydrochloride (purity 99%) was supplied by Toronto Research Chemicals Inc. (Toronto, Canada), and rimantadine-d4 hydrochloride (purity 98%) was obtained from C/D/N Isotopes Inc. (Pointe-Claire, Canada).

HPLC-grade acetonitrile (ACN) and methanol were purchased from Fisher Scientific (Fair Lawn, USA). HPLC-grade formic acid was obtained from Sigma Aldrich (St. Louis, USA). Analytical-grade hydrochloric acid, ammonium hydroxide, and acetic acid were supplied by Beijing Chemical Reagent Co. Ltd (Beijing, China). Ultra-pure water was prepared using a Milli Q-plus system (Billerica, MA, USA). The MCX and PCX SPE column were purchased from Waters (Milford, USA) and Agela Technology (Tianjin, China), respectively.

The feed selected for this experiment included formula feed for pig, premix feed for chicken, complicated concentrated feed for pig were supplied by livestock farms and feed mills in Chinese mainland.

Standard preparation

Stock solutions (1000 mg/L) of targeted analytes (amantadine and rimantadine) and their isotopic internal standards (amantadine-d15 and rimantadine-d4) were individually prepared by dissolving appropriate amount of standards in methanol, and stored in the dark at − 20 °C. The mixed working solution (I) were obtained by diluting stock solutions of two analytes in the same volumetric flask using 50% acetonitrile in water, the working solution (II) of internal standards (IS) at concentration of 1.0 mg/L was also prepared in another volumetric flask with same method. All working solutions were stored at 4 °C. After investigating stability of the mixed working solutions (2, 10, and 100 μg/L) for 14 days, the results showed the relative standard deviations (RSDs) of responses among 0, 7, and 14 days were < 1.98% for targeted analytes during this period. Thus, there is no obvious degradation for the mixed working solutions and they are valid for at least 14 days at 4 °C.

Sample preparation

All feed samples were finely grinded and homogenized using a grinding mill, and stored in – 20 °C. Feed (2 g) spiked in 20 μL of IS working solution (II) was extracted with 10 mL of 1% acetic acid in acetonitrile by vortex for 1 min. After oscillating 30 min and centrifuging at 10000 r/min for 10 min, the supernatant was transferred into a 50-mL centrifuge tube. The sample was extracted again with 10 mL of 1% acetic acid in acetonitrile by vortex for 2 min and the supernatants were combined after centrifugation. Then supernatants were passed through MCX SPE column that were previously processed with 3 mL of methanol and 3 mL of water. Impurities were washed by 3 mL of 2% hydrochloric acid in water and 3 mL of methanol successively. The targeted analytes were eluted by 5 mL of 5% ammonium hydroxide in methanol after being dried for 3 min under vacuum. The elution was concentrated to dry with nitrogen under 50 °C. Finally the residue was redissolved by 1 mL of 50% acetonitrile in water. The spiked feed samples at same concentration were three replicates for method optimization and prepared by adding a suitable volume of the mixed working solution (I) in the blank formula feed for pig.

Apparatus and chromatographic conditions

In the part of liquid chromatography, a SCIEX Exion LC system equipped with a degasser, dual pump, column compartment, autosampler (Wilmington, DE, USA) was used. Chromatographic separations were performed on a XDB-C18 column (2.1 mm × 150 mm, 3.5 μm particle size) from Agilent at a flow rate of 0.3 mL/min in room temperature. The mobile phase consisted of eluent A (0.1% volume ratio of formic acid in water) and eluent B (methanol). Gradient elution began from 90% eluent A, linearly decreased down to 10% in 2 min, held constant for 3 min, then returned to the initial ratio in 0.1 min, and equilibrated for 5 min. The total cycle time was 10 min. Injection volume was set as 5 μL.

Mass spectrometric analysis was achieved by using a QTRAP 6500+ triple quadrupole linear ion trap mass spectrometry (SCIEX, USA). A voltage of 5.0 kV with positive electrospray and source temperature of 500 °C were set. Nitrogen was used as the collision gas. The operational software for data collection and analysis were Analyst 1.6.3. MRM parameters for target drugs and IS are summarized in Table 1.
Table 1

Multi-reaction monitoring parameters for amantadine and rimantadine


MRM ion pair (m/z)

Declustering potential (DP)/V

Collision energy (CE)/eV


152.0 > 135.0a



152.0 > 93.0




167.3 > 150.3a




180.2 > 163.2a



180.2 > 81.0




184.2 > 167.0a



aIon pair for quantification

Aim to obtain more accurate qualitative results, the information-dependent acquisition (IDA) was used in this method. The acquisitions of one ion with a peak height exceeding 500 counts per second were set as its parameters without exclusion after dynamic background subtraction of the survey scan. The enhanced product ion (EPI) spectrum at speed of 10,000 Da/s from m/z = 50 to 200 were used to improve confidence in compound identification, with a dynamic fill time for optimal MS/MS quality. EPI spectrums were generated by using three standardized CE of 20, 35, and 50 eV with a collision energy spread (CES) of 15 eV. This can obtain a characteristic MS/MS pattern that did not depend on the efficiency of the compound fragmentation. EPI spectrums of amantadine and rimantadine and their relevant information including chemical names, molecular formulas, molecular weights, molecular structures, and CAS number were imported database to create a standard library. To obtain the matching results were obtained by searching MS/MS spectrums against the library to compare the unknown data with standard spectrums.

Method validation

A series of working standard solutions with IS concentrations of 20 μg/L were prepared with concentrations of 1, 2, 4, 10, 20, 40, 100, and 200 μg/L for both of amantadine and rimantadine. Calibration curves were plotted the ratio of peak areas to the ratio of concentrations between targeted analytes and internal standards, respectively. The slope ratios of the matrix-matched standard calibration to solvent standard solution calibration were used to investigate the matrix effect. A series of the matrix-matched standard solutions were prepared by diluting the mixed working solution (I) using blank cleanup solutions that were obtained by preparing blank formula feed for pig using above sample preparation method. The limits of detection (LODs) and limits of quantification (LOQs) were used to evaluate the sensitivity of the developed method. The LODs were defined as spiked concentrations that produced the signal-to-noise ratio (S/N) of 3, and LOQs as spiked concentrations with S/N of 10 under the acceptable accuracy and precision. The recoveries of two drugs with five replicates at four spiked concentration levels in different feeds were used to estimate the accuracy and precision. Appropriate volumes of working standard solutions were added in each blank matrix to prepare the spiked samples. Aim to obtain sufficient stability, all spiked samples were set for 30 min after vortex for 30 s. The repeatabilities were evaluated with the intra-day and inter-day relative standard deviations (RSDs).

Results and discussion

Optimization of sample extraction

Different solutions including ACN, 1% acetic acid in ACN, 2% acetic acid in ACN, 5% acetic acid in ACN, acetic acid/ACN/water (1/50/49, v/v/v), and 1% formic acid in ACN were investigated to extract amantadine and rimantadine in feed by shaking. A typical spiked concentration of 20 μg/kg was selected for method optimization, according to pre-experimental results about concentration ranges of calibration curves and our previous report about simultaneous determination of amantadine, rimantadine, and chlorpheniramine in animal-derived food by LC-MS/MS [14]. The experimental results are presented in Fig. 1. When using ACN as extraction solution, the recoveries of amantadine and rimantadine were poor compared to ACN containing acid. The 1% acetic acid in ACN showed higher recoveries and S/N ratios than other solutions containing different contents of acid. Additionally, more interferences and lower S/N ratios were observed when using acetic acid/ACN/water (1/50/49, v/v/v). This phenomenon may be caused by the water in extraction solution that can dissolve more impurities such as hydrophilic compounds etc. Consequently, 1% acetic acid in ACN was selected and used for subsequent experiments.
Fig. 1

The recoveries and S/N ratios of amantadine and rimantadine in spiked feed at 20 μg/kg using different extraction solutions including (a) ACN, (b) 1% acetic acid in ACN, (c) acetic acid/ACN/water (1/50/49, v/v/v), (d) 1% formic acid in ACN, (e) 2% acetic acid in ACN, and (f) 5% acetic acid in ACN

The different volumes (10 and 20 mL) of extraction solution were then investigated, and the results were shown in Fig. 2. The highest recoveries of amantadine and rimantadine in feed were obtained by using 10 mL of 1% acetic acid in ACN and twice extractions. The recoveries were the lowest when only using 10 mL at once extract compared to 20 mL at once extract and 10 mL at twice extracts. Although the average recoveries using internal standard method were > 90% for both analytes, the average recoveries using external standard method obviously increased from 35.8 to 58.2% for amantadine, and from 42.1 to 65.7% for rimantadine with once extract to twice extracts. These indicate that the bigger volume of extraction solution would enhance the recoveries for both analytes but the highest extraction efficiency can be obtained by multiple extracts. Thus, the sample was extracted by using 10 mL of 1% acetic acid in ACN for twice by shaking for 30 min.
Fig. 2

The recoveries of amantadine and rimantadine in spiked feed at 20 μg/kg using internal and external methods with different extraction volumes and times

Optimization of the purification methods

The QuEChERs method was reported for cleanup of antiviral drugs in animal-derived food in our previous study. Firstly, this cleanup procedure was tried for purification of amantadine and rimantadine in feed sample. However, the poor results were obtained based on more interference, asymmetric shapes, lower recoveries, and sensitivity because the feed is more complicated compared to animal-derived food. A SPE cleanup had to be selected for the following sample purification. According to the polarity of amantadine and rimantadine, two types of cation exchange SPE columns including MCX and PCX (60 mg, 3 mL) were investigated. After washing with 3 mL of 2% HCl in water and 3 mL of methanol, the analytes on SPE columns were comparatively eluted by 5% ammonia in methanol and ammonia/methanol/2-propanol (5/70/25, v/v/v). The results in Fig. 3 showed that both of MCX and PCX exhibited good recoveries (> 80%) but MCX exhibited higher recoveries and less interference than PCX. Additionally, 5% ammonia in methanol showed good elution effects for both drugs but ammonia/methanol/2-propanol presented lower recovery of 72.3% for amantadine comparatively. Therefore, MCX column with elution solution of 5% ammonia in methanol was selected for the development of this method.
Fig. 3

The recoveries of amantadine and rimantadine in spiked feed at 20 μg/kg using different sample cleanup methods including (a) MCX and (b) PCX columns with ammonia/methanol (5/95, v/v) and (c) MCX column with ammonia/methanol/2-propanol (5/70/25, v/v/v)

Choice of redissolved solvent

There are different influences on the peak shape and response of target analytes in UHPLC-MS/MS. So, the redissolved solvents including ACN, methanol, ACN/water (50/50, v/v), and methanol/water (50/50, v/v) were selected for optimization. The results showed that 100% ACN would lead to asymmetric peak shapes and lower responses, and methanol had higher noise because it has higher polarity and would dissolve more interferences. Comparatively, ACN/water (50/50, v/v) exhibited good peak shapes and responses, and had better cleanup effects because of its lower solubility for protein and fat compared to methanol/water. Thus, it enabled higher UHPLC–MS/MS sensitivity for the determination of amantadine and rimantadine in feed.

Matrix effect

Matrix effects play an important role in the quality of the quantitative data generated by a method because some co-eluted components can suppress or enhance response signals of target analytes [26]. The slope ratios of two drugs were given in Table 2. The results showed that the slope ratios (0.97 and 1.06) were within ± 10% of the slope ratio of 1.0, indicates the matrix effect of this method was negligible. This may be contributed to both of the better cleanup effect by SPE column and the calibration of the internal standard method that can effectively decline the matrix effect [12].
Table 2

Regression data and sensitivity of the developed method


Linear equation

Concentration range (μg/L)


Matrix effecta

LOD (μg/kg)

LOQ (μg/kg)


y = 1.08x + 0.0176







y = 1.83x + 0.00972






aMatrix effect is the slope ratio using the internal standard method

Simultaneous quantitation and qualitative confirmation by MRM-IDA-EPI mode

A UHPLC-Qtrap-MS was used in the developing of this method. In the IDA experiment, this system can using MRM-IDA-EPI mode to carry out quantitative MRMs and qualitative QTRAP scans in the same injection, and thus can simultaneously obtain quantitative and qualitative results. Therefore, a MRM transition spectrum at the correct retention time, and the matching results of molecular and fragment ion data from the library were set as the identification criteria of targeted analytes in samples. The correlation between spectrums is typically expressed as the fit value that reflects the similarity between the reference and the unknown spectrum, the reverse fit value that reflects the similarity between the unknown and the reference spectrum, and the purity fit value that is a combination of the previous two values. The last one generally used for quantification and 70% had been set as an appropriate threshold for the positive identification of the analytes in samples by the instrument manufacturer [22]. According to this, a series of the standard solutions with concentrations from 0.05 to 200 μg/L were injected into instrument using MRM-IDA-EPI mode. The results showed that enough purity fit values can be obtained until lower concentration of 0.2 μg/L for both of amantadine and rimantadine, indicates better qualitative ability and effective improvement of method reliability compared to traditional MRM mode. Moreover, the higher sensitivity for both analytes was also obtained even using MRM-IDA-EPI mode, indicates this mode would not obviously affect analytical sensitivity for both drugs. However, the LODs and LOQs of this method cannot be directly set based on these results, as the acceptable recoveries, precisions, and S/N ratio should be considered by investigation using spiked feed samples at the same time.

Linearity and sensitivity

The linearity of the developing method was investigated by calibration curves with internal standard method. The curves for two analytes were constructed with a linear regression with 1/x weighting were partly shown in Table 2. Good linearities with relative coefficients (R) higher than 0.9996 were obtained over the concentration range of 1–200 μg/L. The k value of amantadine was 1.08 and smaller than that (1.83) of rimantadine, indicates that the responses of amantadine were more positively proportional to its concentrations compared to that of rimantadine. The LODs and LOQs were 0.2 and 0.5 μg/kg respectively for both of amantadine and rimantadine because of the acceptable S/N ratios (> 3) at 0.2 μg/kg and average recoveries (90.7–103%), precisions (3.52–8.52%), S/N ratios (> 10) at 0.5 μg/kg (Fig. 4). This suggests significantly higher sensitivity than previous report that used UHPLC-MS/MS coupled with MCX SPE column to detect amantadine in animal tissues and obtained LODs of 5.0 μg/kg [2]. This sensitivity also is similar with that in our previous study for determination of amantadine and rimantadine in animal-derived food using QuEChERs method to cleanup. Additionally, the concentrations of standard solutions (0.4 μg/L) at LODs were higher than the lowest concentrations (0.2 μg/L) that can be effectively qualitatively confirmed using above MRM-IDA-EPI mode, indicates that the developed method has better qualitative ability. To our knowledge, few reports involved in the detection of these drugs in feed until now although there are some references about their detections in pharmaceutical formulations [27], human [28] and animal plasma [29, 30] and animal-derived food [14, 16, 23]. Therefore, the developed method will firstly provide an important and sensitive measure to monitor their presences in feed.
Fig. 4

The typical chromatograms of amantadine and rimantadine in spiked feed at 0.5 μg/kg

Accuracy and precision

The accuracy and precision of the developed method were described by intra- and inter-day variability assays at four spiked levels of 0.5, 2, 10, and 100 μg/kg for amantadine and rimantadine. Table 3 summarizes an overview of recoveries and RSDs of two analytes in three feed matrices including formula feed for pig, premix feed for chicken, and complex concentrated feed for pig. The recoveries ranged from 76.1 to 112% with intra-day RSDs within 9.61%, and inter-day RSDs within 8.46%, indicates good accuracy and precision (RSD ≤ 10%) for the method.
Table 3

Intra-day and inter-day precisions for two analytes in feed (n = 5)


Spiked level (μg/kg)











Formula feed for pig


93.5 ± 7.97

93.8 ± 6.59

91.3 ± 2.81

90.7 ± 3.40


108 ± 6.22

107 ± 6.08

99.4 ± 9.25

98.4 ± 7.72


79.9 ± 5.71

80.8 ± 5.00

76.1 ± 6.82

77.9 ± 6.51


95.3 ± 3.69

94.5 ± 3.35

87.8 ± 3.10

89.9 ± 4.67

Premix feed for chicken


98.6 ± 3.47

96.8 ± 4.19

100 ± 7.40

100 ± 6.54


108 ± 9.49

106 ± 8.45

104 ± 9.31

105 ± 7.99


86.7 ± 9.26

88.3 ± 8.05

86.1 ± 9.61

87.9 ± 8.46


87.5 ± 4.74

89.5 ± 5.14

96.7 ± 4.94

97.1 ± 4.15

Complex concentrated feed for pig


103 ± 6.74

101 ± 6.29

95.8 ± 3.48

94.3 ± 5.37


112 ± 5.94

109 ± 6.67

102 ± 4.54

103 ± 5.12


102 ± 9.49

100 ± 8.41

92.2 ± 8.78

91.5 ± 7.37


95.0 ± 4.24

96.5 ± 4.53

91.6 ± 4.46

92.4 ± 4.79

The value is the average recovery ± SD

Application to real sample

The method described above was practically applied to the simultaneous determination of amantadine and rimantadine in 132 feed samples obtained from commercial origin in China. Firstly, a QuEChERS method with extraction of ACN/water (50/50, v/v) and fast cleanup with primary secondary amine (PSA) sorbent (50 mg of PSA to 1 mL of extraction solution) was used to obtain a fast screening results of two analytes. The initial quantitative results using traditional MRM ion pairs showed that suspected amantadine were observed in all samples because of its presences of typical chromatographic peaks and similar MRM ion pairs (Fig. 5), and no rimantadine was found. However, after using the developed instrumental method (UHPLC-Qtrap-MS based on MRM-IDA-EPI mode) to analyze the matching results of molecular and fragment ion data from the library, no positive results for amantadine were found according to lower fit value of 31–56% for amantadine in real samples. For example, as Fig. 5 shown, the typical mass spectrum of amantadine in standard solution has similar ions of m/z 153 and 135 with that in real suspected feed sample. But different ion composition and abundances (m/z 134, 106, and 58 etc.) existed in processed feed samples using QuEChERS method. Secondly, all 132 samples were analyzed again to obtain more accurate quantitative results using the developed SPE method. The results showed that both of amantadine and rimantadine have not been detected in all samples. As Fig. 6 shown, the chromatographic peaks of amantadine and other compounds were not observed at the corresponding retention times, although a suspected peak of amantadine presented on the MRM chromatogram from QuEChERS method. Furthermore, the characteristic spectrums also have not been obtained using UHPLC-Qtrap-MS based on MRM-IDA-EPI mode. These results indicate that the developed method has better sample cleanup effects and can effectively eliminate some impurities in feed compared to fast screening method, and presented more accurate quantitative and qualitative abilities.
Fig. 5

The comparison of typical mass spectrums for amantadine in (a) standard solution and in (b) a suspected feed sample

Fig. 6

The comparison of typical MRM chromatograms of amantadine in a suspected feed sample using (a) the fast QuEChERs method and (b) the develop method with SPE cleanup


In this study, a new UHPLC-QTRAP-MS method was developed and applied for simultaneous determination of amantadine and rimantadine in feed. After optimization of sample preparation, MCX SPE column was used for sample cleanup, and then MRM-IDA-EPI mode was used for simultaneous quantitative determination and qualitative confirmation of these two drugs. The results from assay validation showed that the developed method has good accuracy, precision, and sensitivity. Its application in detection of real feed samples showed that the MRM-IDA-EPI mode can effectively improve the reliability even at a lower concentration compared to traditional MRM mode. The developed method provides a sensitive and reliable tool for monitoring amantadine and rimantadine in feed for law enforcement purpose.



This work was supported by Central Public-interest Scientific Institution Basal Research Fund (Nos. Y2017JC46 and 1610072016003) and the Special Fund for Agro-scientific Research in the Public Interest (201203023).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.


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Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  • Qi Jia
    • 1
  • Dan Li
    • 1
  • Xinlu Wang
    • 1
  • Shuming Yang
    • 1
  • Yongzhong Qian
    • 1
  • Jing Qiu
    • 1
  1. 1.Institute of Quality Standards & Testing Technology for Agro-Products, Key Laboratory of Agro-product Quality and SafetyChinese Academy of Agricultural Sciences; Key Laboratory of Agri-food Quality and Safety, Ministry of AgricultureBeijingChina

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