Determination of amino glycosides in honey by capillary
electrophoresis tandem mass spectrometry and extraction with
molecularly imprin ted polymers
David Moreno-Gonzalez a, Francisco J. Lara a, Nikola Jurgovska b, Laura Gamiz-Gracia a,
Ana M. García-Campana~ a, *
a Department of Analytical Chemistry, Faculty of Sciences, University of Granada, Av. Fuentenueva s/n, E-18071 Granada, Spain
b Charles University, Faculty of Pharmacy in Hradec Kralov e, Prague, Checz Republic
h i g h l i g h t s g r a p h i c a l a b s t r a c t
CE-MS/MS proposed the first time for
the determination of 9 aminoglycosides in honeys.
Molecularly imprinted polymer for
the selective solid phase extraction of
analytes.
High sensitivity with limits of quantification from 1.4 to 94.8 mg kg 1.
Recoveries ranged from 88.2 to 99.8%,
with RSD lower than 8%.
a r t i c l e i n f o
Article history:
Received 24 March 2015
Received in revised form
6 August 2015
Accepted 10 August 2015
Available online xxx
Keywords:
Aminoglycosides
Capillary zone electrophoresis
Tandem mass spectrometry
Stacking
Molecularly imprinted polymers
Honey
a b s t r a c t
A new analytical method based on capillary zone electrophoresis-tandem mass spectrometry is proposed
and validated for the identification and simultaneous quantification of nine aminoglycosides in honey
samples. Detection using an ion trap mass analyzer operating in the multiple reaction monitoring mode
was used. Different parameters were optimized in order to obtain an adequate separation combined with
the highest sensitivity. In order to achieve high selectivity in the sample treatment, a commerciallyavailable molecularly imprinted polymer has been used for the solid phase extraction of the analytes.
Under optimum conditions, recoveries for fortified samples ranged from 88.2 to 99.8%, with relative
standard deviations lower than 8%. The limits of detection ranged from 0.4 to 28.5 mg kg 1. Furthermore,
the decision limit and the detection capability were evaluated, ranging from 3.5 to 60.5 mg kg 1 and from
6.0 to 103.1 mg kg 1, respectively, demonstrating the sensitivity and applicability of this fast and simple
method.
© 2015 Elsevier B.V. All rights reserved.
Abbreviations: AG, Aminoglycoside; APM, Apramycin; BGE, Background electrolyte; CZE, Capillary zone electrophoresis; CCa, Decision limit; CCb, Detection capability;
DHS, Dihydrostreptomycin; EU, European Union; FASS, Field-amplified sample stacking; GENT, Gentamicin; HFBA, Heptafluorobutyric acid; HILIC, Hydrophilic interaction
chromatography; IT, Ion trap; MRLs, Maximum residue limits; MeOH, Methanol; MIPs, Molecularly imprinted polymers; MRM, Multiple reaction monitoring; NEO,
Neomycin; PRM, Paromomycin; IPA, Isopropanol; RASFF, Rapid Alert System for Food and Feed; SPE, Solid phase extraction; MISPE, Solid phase extraction with molecularly
imprinted polymers; SPC, Spectinomycin; STP, Streptomycin.
* Corresponding author.
E-mail address: amgarcia@ugr.es (A.M. García-Campana).~
Contents lists available at ScienceDirect
Analytica Chimica Acta
journal homepage: www.elsevier.com /locate/aca
http://dx.doi.org/10.1016/j.aca.2015.08.003
0003-2670/© 2015 Elsevier B.V. All rights reserved.
Analytica Chimica Acta xxx (2015) 1e8
Please cite this article in press as: D. Moreno-Gonzalez, et al., Determination of aminoglycosides in honey by capillary electrophoresis tandem
mass spectrometry and extraction with molecularly imprinted polymers, Analytica Chimica Acta (2015), http://dx.doi.org/10.1016/
j.aca.2015.08.003
1. Introduction
Aminoglycoside (AG) antibiotics are widely used in veterinary
medicine. They are commonly used to treat foulbrood infection
(caused by bacteria), and Nosema disease (caused by protozoa).
Veterinary use of antibiotics is regulated by European Union (EU)
and maximum residue limits (MRLs) have been established in
different edible parts or products derived from animals, such as
muscle, kidney, fat, liver, milk, and eggs [1]. EU does not authorize
the use of antibiotics, including AGs, in beekeeping and MRLs have
not been established for these compounds in honey, applying the
so-called “zero tolerance”. However, honey and products derived
from bees coming from non-EU countries, might be contaminated
with AG residues, which is a major concern in the honey trade [2,3].
Some studies have revealed that a substantial part of the currently
marketed honey contains residues of antibiotics [4] and since 2003,
the EU's Rapid Alert System for Food and Feed (RASFF) has regularly
alerted Member States about the presence of antibiotic residues in
these products [5].
The EU Community Reference Laboratories for veterinary residues have established “action limits” for the validation of analytical
methods in relation to the control of unauthorised analytes in
different matrixes [6]. In relation to AGs in honey, a recommended
concentration of 40 mg kg 1 has been set for streptomycin (SPT),
which involves that detection capability (CCb) for screening
methods or decision limit (CCa) for confirmatory methods should
be lower than this value.
Traditionally, high performance liquid chromatography (HPLC)
methods have been applied for the determination of AGs [7],
mainly using as detection system tandem mass spectrometry (MS/
MS) with positive electrospray ionization mode (ESI þ). However,
the high polarity of these antibiotics is a drawback for their
analysis by HPLC, as they are not retained in reverse-phase columns. Ion-pair chromatography has been proposed as an alternative to obtain a satisfactory separation of these compounds
[8e12]. Nevertheless, ion-pair chromatography requires the addition of an ion-pair reagent (mainly trifluoroacetic acid or heptafluorobutyric acid) in the mobile phase. These ion-pair reagents
are rarely volatile acids and can seriously affect the performance of
MS, causing ionization suppression of analytes and contamination
of the ion source. Hydrophilic interaction chromatography (HILIC)
has been recently proposed as an alternative to ion-pair chromatography for the analysis of AGs in honey by LC-MS [11,13e16].
However, in this methodology, a high concentration of salts in the
mobile phase is usually needed which can be detrimental for MS
detection.
Capillary electrophoresis (CE) can be an interesting alternative
to HPLC for the analysis of these compounds due to its advantages
such as short analysis time, high separation efficiency and low reagent consumption. A drawback is the absence of chromophore
groups in AGs, which prevents their determination by UV/Vis, the
most common detection method in CE. Several attempts have been
made to overcome this problem, i.e. the use of indirect UV detection
[17,18] or the application of derivatization methods to form absorbent species in the UV/Vis region [19]. Also post-column derivatization was used to apply laser induced fluorescence detection [20].
Unfortunately, these methods show poor sensitivity because of the
short optical path length and the small volume of sample injected
or involve tedious and complicated derivatization processes.
MS/MS is an alternative to improve sensitivity and selectivity in
CE, allowing the unequivocal identification of antibiotic residues
and therefore fulfilling EU regulation requirements [21]. Unfortunately, the small volume of sample injected in CE continues to be a
major constraint. In order to mitigate this problem numerous
strategies have been developed [22e24]. Among them, fieldamplified sample stacking (FASS) is one of the most effective and
simplest methods to achieve high sensitivity [25]. In FASS, the
sample solvent has conductivity lower than the background electrolyte (BGE). Therefore, when a voltage is applied, the electric field
will be higher in the sample zone than in the BGE. As a result,
analytes migrate quickly in the sample zone and slow down when
they reach the BGE, causing the “stacking” of the analytes around
the sample-BGE boundary.
Another issue that needs to be solved in the determination of
AGs is the sample preparation. It is not an easy task due to the high
polarity of AGs and their tendency to bind strongly to matrix
proteins. Most of the methods reported for the analysis of AGs in
honey involve the use of solid phase extraction (SPE) cartridges,
such as weak cation exchange [14,15], octadecyl [12] or
hydrophilic-lipophilic balance [9,16] to ensure a clean extract.
Other materials with higher selectivity, such as molecularly
imprinted polymers (MIPs) can provide cleaner extracts. MIPs are
synthetic materials with artificially generated recognition sites
able to specifically capture target molecules [26,27]. Thus, the
strong interaction between MIPs and target molecules makes
them ideal for the selective extraction of compounds at trace
levels, particularly when the sample is complex. Several reviews
show their applications in analytical chemistry [28e30]. Specifically, the use of MIPs as SPE sorbents (MISPE) for the selective
extraction of antibiotics from food samples has grown significantly
in the last few years [31e35]. Recently, Ji et al. have synthesized a
MIP sorbent for AGs using SPT as the template molecule, obtaining
satisfactory efficiency and selectivity in the analysis of honey
samples [13].
In this work, we propose a useful alternative to quantify very
low concentrations of nine AGs in honey using a recently
commercially available MIPs. The use of CE-MS/MS with FASS
preconcentration is also proposed as an approach for improving
sensitivity and selectivity. The obtained results demonstrate for the
first time the possibilities of MISPE and CE-MS/MS for the quantification of AG residues in honey.
2. Experimental
2.1. Reagents and materials
It has to be highlighted that, because of the high sorption affinity
of the AGs to polar surfaces and their high photosensitivity, polypropylene amber vessels (flasks, glass and vials) were used during
sample preparation, storage and injection.
Ultrapure water
ความมุ่งมั่นของ glycosides อะมิโนในน้ำผึ้งโดยหลอดเลือดฝอยelectrophoresis ตัวตามกันไปรเมทและการสกัดด้วยmolecularly imprin เท็ดโพลิเมอร์David Moreno Gonzalez Lara J. Francisco a, a, b Jurgovska สเวตินิโกลา ลอร่า Gamiz Gracia เป็นเอเอ็นเอม. García Campana ~ a, *ภาควิชาเคมีวิเคราะห์ คณะวิทยาศาสตร์ มหาวิทยาลัยกรานาดา Av. Fuentenueva s/n, E-18071 กรานาดา ประเทศสเปนมหาวิทยาลัยชาร์ลส์บี คณะเภสัชศาสตร์ใน Hradec Kralov e ปราก Checz สาธารณรัฐh ฉัน g h l ฉัน g h t s g r p เป็น h ฉัน c l กับ b s t r c tครั้งแรกสำหรับการนำเสนอ CE-MS/MSกำหนด 9 aminoglycosides ใน honeysพอลิเมอร์ที่ติดตรา molecularly สำหรับการสกัดเลือกเฟสของแข็งanalytesความไวที่สูงกับขีดจำกัดของนับจาก 1.4 การมก. 94.8 กก. 1Recoveries อยู่ในช่วงจาก 88.2 99.8%มีต่ำกว่า 8% RSDr t ฉัน c l e ฉัน n f oบทความประวัติ:ได้รับ 24 2015 มีนาคมได้รับในแบบฟอร์มการปรับปรุง6 2015 สิงหาคมยอมรับ 10 2015 สิงหาคมXxx ออนไลน์พร้อมใช้งานคำสำคัญ:AminoglycosidesElectrophoresis โซนเส้นเลือดฝอยตัวตามกันไปรเมทซ้อนติดตรา molecularly โพลิเมอร์น้ำผึ้งแบบ b s t r c tมีเสนอวิธีวิเคราะห์ใหม่ตามรูพรุนโซน electrophoresis ตัวตามกันไปรเมทและตรวจนับพร้อมของเก้า aminoglycosides ในน้ำผึ้งและรหัสตัวอย่างการ ตรวจสอบโดยใช้การตรวจจับไอออนมวลในปฏิกิริยาหลายโหมดการตรวจสอบวิเคราะห์ใช้ พารามิเตอร์ต่าง ๆ ถูกปรับให้เหมาะสมเพื่อให้ได้การแยกพอรวมกับระดับความสำคัญสูงสุด เพื่อให้บรรลุใวสูงในการรักษาตัวอย่าง เมอร์ molecularly ติดตรา commerciallyavailable มีการใช้การสกัดของแข็งเฟสของ analytesภายใต้เงื่อนไขที่เหมาะสม recoveries สำหรับตัวอย่างธาตุที่อยู่ในช่วงจาก 88.2 99.8% มีญาติส่วนเบี่ยงเบนมาตรฐานต่ำกว่า 8% ข้อจำกัดของการตรวจสอบที่อยู่ในช่วงจาก 0.4 ถึง 28.5 มิลลิกรัมกิโลกรัม 1 นอกจากนี้จำกัดการตัดสินใจและสามารถตรวจสอบได้ประเมิน ตั้งแต่ 3.5 มก. 60.5 กก. 1 และจาก6.0-103.1 มก.กก. 1 ตามลำดับ เห็นความไวและความเกี่ยวข้องของของง่าย และรวดเร็วนี้วิธีการ© 2015 Elsevier b.v สงวนลิขสิทธิ์ทั้งหมดตัวย่อ: AG, Aminoglycoside APM, Apramycin BGE อิเล็กโทรพื้น CZE แรงโซน electrophoresis CCa ขีดจำกัดตัดสินใจ CCb ความสามารถในการตรวจสอบDHS, Dihydrostreptomycin ยุโรป สหภาพยุโรป FASS ขยายฟิลด์อย่างซ้อน GENT, Gentamicin HFBA, Heptafluorobutyric กรด HILIC, Hydrophilic โต้ตอบchromatography มัน จับไอออน MRLs สูงสุดตกค้างจำกัด ทานอ เมทานอล MIPs, Molecularly ติดตราโพลิเมอร์ MRM ปฏิกิริยาหลายตรวจสอบ นีโอนีโอมัยซิน PRM, Paromomycin IPA, Isopropanol RASFF ระบบแจ้งเตือนอย่างรวดเร็วในอาหารและอาหาร SPE การแยกเฟสของแข็ง MISPE ทึบระยะสกัดกับ molecularlyติดตราโพลิเมอร์ SPC, Spectinomycin STP, Streptomycin* ผู้สอดคล้องกันที่อยู่อีเมล์: amgarcia@ugr.es (น. García Campana) . ~เนื้อหารายการ ScienceDirectคตา Analytica Chimicaหน้าแรกของสมุดรายวัน: www.elsevier.com/ค้น หา/acahttp://dx.doi.org/10.1016/j.aca.2015.08.0030003-2670 / © 2015 Elsevier b.v สงวนลิขสิทธิ์ทั้งหมด1e8 Analytica Chimica คตา xxx (2015)กรุณาอ้างอิงบทความนี้ในข่าวเป็น: D. Moreno Gonzalez, et al. กำหนด aminoglycosides ในน้ำผึ้งโดย electrophoresis รูพรุนตัวตามกันไปmass spectrometry และสกัด ด้วย molecularly ติดตราโพลิเมอร์ Analytica Chimica คตา (2015), http://dx.doi.org/10.1016/j.aca.2015.08.0031. บทนำยา Aminoglycoside (AG) ใช้ในสัตวยา พวกเขามักจะใช้การรักษาติดเชื้อ foulbrood(เกิดจากแบคทีเรีย), Nosema โรค (โพรโทซัว) และการการใช้ยาปฏิชีวนะที่สัตวแพทย์กำหนดโดยสหภาพยุโรป (EU)และมีการสร้างสูงสุดตกค้างจำกัด (MRLs) ในส่วนการกินต่าง ๆ หรือผลิตภัณฑ์ได้มาจากสัตว์ เช่นกล้ามเนื้อ โรคไต ไขมัน ตับ นม และไข่ [1] EU ไม่อนุญาตมีการใช้ยาปฏิชีวนะ AGs รวมทั้งใน beekeeping และ MRLsไม่สร้างสารเหล่านี้ในน้ำผึ้ง ใช้การเรียกว่า "ศูนย์ยอมรับ" อย่างไรก็ตาม ที่มาของน้ำผึ้งและผลิตภัณฑ์จากผึ้งที่มาจากประเทศ EU ไม่ อาจจะปนเปื้อนมีตก AG ซึ่งเป็นเรื่องสำคัญในการค้าขายน้ำผึ้ง [2,3]Some studies have revealed that a substantial part of the currentlymarketed honey contains residues of antibiotics [4] and since 2003,the EU's Rapid Alert System for Food and Feed (RASFF) has regularlyalerted Member States about the presence of antibiotic residues inthese products [5].The EU Community Reference Laboratories for veterinary residues have established “action limits” for the validation of analyticalmethods in relation to the control of unauthorised analytes indifferent matrixes [6]. In relation to AGs in honey, a recommendedconcentration of 40 mg kg 1 has been set for streptomycin (SPT),which involves that detection capability (CCb) for screeningmethods or decision limit (CCa) for confirmatory methods shouldbe lower than this value.Traditionally, high performance liquid chromatography (HPLC)methods have been applied for the determination of AGs [7],mainly using as detection system tandem mass spectrometry (MS/MS) with positive electrospray ionization mode (ESI þ). However,the high polarity of these antibiotics is a drawback for theiranalysis by HPLC, as they are not retained in reverse-phase columns. Ion-pair chromatography has been proposed as an alternative to obtain a satisfactory separation of these compounds[8e12]. Nevertheless, ion-pair chromatography requires the addition of an ion-pair reagent (mainly trifluoroacetic acid or heptafluorobutyric acid) in the mobile phase. These ion-pair reagentsare rarely volatile acids and can seriously affect the performance of
MS, causing ionization suppression of analytes and contamination
of the ion source. Hydrophilic interaction chromatography (HILIC)
has been recently proposed as an alternative to ion-pair chromatography for the analysis of AGs in honey by LC-MS [11,13e16].
However, in this methodology, a high concentration of salts in the
mobile phase is usually needed which can be detrimental for MS
detection.
Capillary electrophoresis (CE) can be an interesting alternative
to HPLC for the analysis of these compounds due to its advantages
such as short analysis time, high separation efficiency and low reagent consumption. A drawback is the absence of chromophore
groups in AGs, which prevents their determination by UV/Vis, the
most common detection method in CE. Several attempts have been
made to overcome this problem, i.e. the use of indirect UV detection
[17,18] or the application of derivatization methods to form absorbent species in the UV/Vis region [19]. Also post-column derivatization was used to apply laser induced fluorescence detection [20].
Unfortunately, these methods show poor sensitivity because of the
short optical path length and the small volume of sample injected
or involve tedious and complicated derivatization processes.
MS/MS is an alternative to improve sensitivity and selectivity in
CE, allowing the unequivocal identification of antibiotic residues
and therefore fulfilling EU regulation requirements [21]. Unfortunately, the small volume of sample injected in CE continues to be a
major constraint. In order to mitigate this problem numerous
strategies have been developed [22e24]. Among them, fieldamplified sample stacking (FASS) is one of the most effective and
simplest methods to achieve high sensitivity [25]. In FASS, the
sample solvent has conductivity lower than the background electrolyte (BGE). Therefore, when a voltage is applied, the electric field
will be higher in the sample zone than in the BGE. As a result,
analytes migrate quickly in the sample zone and slow down when
they reach the BGE, causing the “stacking” of the analytes around
the sample-BGE boundary.
Another issue that needs to be solved in the determination of
AGs is the sample preparation. It is not an easy task due to the high
polarity of AGs and their tendency to bind strongly to matrix
proteins. Most of the methods reported for the analysis of AGs in
honey involve the use of solid phase extraction (SPE) cartridges,
such as weak cation exchange [14,15], octadecyl [12] or
hydrophilic-lipophilic balance [9,16] to ensure a clean extract.
Other materials with higher selectivity, such as molecularly
imprinted polymers (MIPs) can provide cleaner extracts. MIPs are
synthetic materials with artificially generated recognition sites
able to specifically capture target molecules [26,27]. Thus, the
strong interaction between MIPs and target molecules makes
them ideal for the selective extraction of compounds at trace
levels, particularly when the sample is complex. Several reviews
show their applications in analytical chemistry [28e30]. Specifically, the use of MIPs as SPE sorbents (MISPE) for the selective
extraction of antibiotics from food samples has grown significantly
in the last few years [31e35]. Recently, Ji et al. have synthesized a
MIP sorbent for AGs using SPT as the template molecule, obtaining
satisfactory efficiency and selectivity in the analysis of honey
samples [13].
In this work, we propose a useful alternative to quantify very
low concentrations of nine AGs in honey using a recently
commercially available MIPs. The use of CE-MS/MS with FASS
preconcentration is also proposed as an approach for improving
sensitivity and selectivity. The obtained results demonstrate for the
first time the possibilities of MISPE and CE-MS/MS for the quantification of AG residues in honey.
2. Experimental
2.1. Reagents and materials
It has to be highlighted that, because of the high sorption affinity
of the AGs to polar surfaces and their high photosensitivity, polypropylene amber vessels (flasks, glass and vials) were used during
sample preparation, storage and injection.
Ultrapure water
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