Hydrophilic interaction chromatography (HILIC) in the analysis of antibiotics

Accepted Manuscript Title: Hydrophilic interaction chromatography (HILIC) in the analysis of antibiotics Author: Getu Kahsay Huiying Song Ann Van Schepdael Deirdre Cabooter Erwin Adams PII: DOI: Reference:

S0731-7085(13)00161-1 http://dx.doi.org/doi:10.1016/j.jpba.2013.04.015 PBA 9037

To appear in:

Journal of Pharmaceutical and Biomedical Analysis

Received date: Revised date: Accepted date:

28-2-2013 10-4-2013 13-4-2013

Please cite this article as: G. Kahsay, H. Song, A. Van Schepdael, D. Cabooter, E. Adams, Hydrophilic interaction chromatography (HILIC) in the analysis of antibiotics, Journal of Pharmaceutical and Biomedical Analysis (2013), http://dx.doi.org/10.1016/j.jpba.2013.04.015 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Hydrophilic interaction chromatography (HILIC) in the analysis

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of antibiotics

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Getu Kahsay, Huiying Song, Ann Van Schepdael, Deirdre Cabooter, Erwin Adams*

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Laboratorium voor Farmaceutische Analyse, Faculteit Farmaceutische Wetenschappen, KU Leuven, O&N2, PB-923, Herestraat 49, B-3000 Leuven, Belgium

ABSTRACT

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This paper presents a general overview of the application of hydrophilic interaction liquid

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chromatography (HILIC) in the analysis of antibiotics in different sample matrices including

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pharmaceutical, plasma, serum, fermentation broths, environmental water, animal origin,

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plant origin, etc. Specific applications of HILIC for analysis of aminoglycosides, β-lactams,

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tetracyclines and other antibiotics are reviewed. HILIC can be used as a valuable alternative

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LC mode for separating small polar compounds. Polar samples usually show good solubility

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in the mobile phase containing some water used in HILIC, which overcomes the drawbacks of

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the poor solubility often encountered in normal phase LC. HILIC is suitable for analyzing

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compounds in complex systems that elute near the void in reversed-phase chromatography.

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Ion pair reagents are not required in HILIC which makes it convenient to couple with MS

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hence its increased popularity in recent years. In this review, the retention mechanism in

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HILIC is briefly discussed and a list of important applications is provided including main

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experimental conditions and a brief summary of the results. The references provide a

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comprehensive overview and insight into the application of HILIC in antibiotics analysis.

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Keywords:

Antibiotics; HILIC; Retention mechanism

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*Corresponding author. Tel: +3216323444; Fax: +3216323448 1

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E-mail address: [email protected]

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Contents

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1. Introduction

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2. Stationary and mobile phases for HILIC application 2.1. Stationary phases in HILIC

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2.2. Mobile phases in HILIC

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3. Use of HILIC for analysis of antibiotics 3.1. Aminoglycoside antibiotics

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3.1.1. Analysis of aminoglycosides

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3.1.2. HILIC in the analysis of aminoglycosides

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3.3. Tetracycline antibiotics

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3.4. Other antibiotics

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References

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3.2. Beta-lactam antibiotics

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1. Introduction Looking back into the history of liquid chromatography (LC), the pioneer systems were

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operated in normal phase mode (NPLC) which implied that the stationary phase was polar.

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Such stationary phase, however, exhibited heterogeneity that resulted in peak tailing and

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non-linear retention factors with varying analyte concentrations [1]. Elution in NPLC has

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been accomplished by non-polar organic solvents. Some variants like the use of isohydric

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solvents were tried, but this approach was found to be rather tedious and failed to yield good

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performance over a longer period [2-4]. The development of stationary phases for

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chromatography driven by pharmaceutical studies of water/octanol partitioning, led to the

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now well-established reversed phase (RP) systems [5, 6]. Bonded octadecyl stationary phases

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allowed efficient separation of analytes within a broad range of polarity and fast column

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equilibration. The lack of retention of highly hydrophilic compounds with ionizable

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functional groups has been supplemented by ion exchange chromatography [7] or ion pairing

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on RP columns [8]. However, for a large group of strongly hydrophilic compounds that could

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not be ionized in solution, it was impossible to obtain retention on either stationary phase. The

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problem has been overcome sometimes in GC by derivatization, and recently, in LC by

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developing hydrophilic interaction chromatography (HILIC) [9].

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The term “hydrophilic” refers to the affinity to water. The concept of HILIC is as follows:

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a polar stationary phase retains polar analytes that are eluted by a mixture of organic solvent

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(usually acetonitrile (ACN)) and water. HILIC is an alternative high-performance liquid

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chromatography (HPLC) mode for separating polar compounds. HILIC has been reported as a

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variant of NPLC, but the separation mechanism in HILIC is more complicated than that in 4

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NPLC [1, 10, 11]. While the acronym HILIC was first suggested by Alpert in 1990 [9], the

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number of publications on HILIC has increased substantially since 2003 as indicated

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elsewhere [11] and as outlined in the review by Hemström and Irgum [12].

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charged basic groups in a solute led to pronounced hydrophilicity and retention. He

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considered that the retention mechanism consisted mostly of partitioning between the bulk

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mobile phase and a layer of mobile phase enriched with water, partially immobilized on the

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stationary phase [9]. Hemström and Irgum [12] concluded that both adsorption and the

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partitioning mechanism contribute to retention in HILIC. Models describing the retention

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mechanism in HILIC can be found in references [10] and [13]. Like NPLC, HILIC employs

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traditional polar stationary phases such as silica, amino or cyano, but the mobile phase used is

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similar to those employed in the RPLC mode [9, 11, 12].

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Alpert noted that

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Originally, HILIC was applied mainly for the determination of carbohydrates, amino

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acids and peptides [10]. Since then, HILIC has been successfully applied to all types of liquid

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chromatographic separations, including small molecules [14], pharmaceutical compounds

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[15], metabolites [16], drugs of abuse [17], toxins [18], carbohydrates [19], oligosaccharides

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[20], amino acids [21], peptides and proteins [9, 22]. The interest in the HILIC technique in

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the last years has been promoted by growing demands for the analysis of polar drugs,

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metabolites and biologically important compounds in proteomics, glycomics and clinical

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analysis [23]. HILIC also allows the analysis of charged substances, as in ion chromatography

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(IC). Another reason for the increasing popularity of HILIC is its excellent suitability for

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coupling to mass spectrometry (MS). Fig. 1 shows how HILIC complements other areas of

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chromatography and extends the range of separation options [11].

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In general, advantages of HILIC have been summarized [24] as follows: (i) good peak

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shapes can be obtained for bases, (ii) MS sensitivity is enhanced due to the high organic

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content in the mobile phase and the high efficiency of spraying and desolvation techniques,

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(iii) direct injection can often be made of extracts eluted from C18 solid-phase extraction

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columns with solvents of high organic content, as these are weak solvents in HILIC, (iv) the

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order of elution of solutes is different and generally the opposite of that found in RP

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separations, (v) good retention of polar compounds is obtained in HILIC, whereas very poor

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retention is often obtained in RPLC and (vi) higher flow rates are possible due to the high

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organic content and hence lower viscosity of typical mobile phases. In addition, polar samples

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always show good solubility in the water containing mobile phase used in HILIC, which

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overcomes the drawbacks of the poor solubility often encountered in NPLC. Ion pair reagents

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are not required in HILIC, and it can be conveniently coupled to MS, especially in the

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electrospray ionization (ESI) mode. In contrast to RPLC, gradient elution HILIC begins with

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a low-polarity organic solvent and elutes polar analytes by increasing the polar aqueous

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content [25].

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Other advantages of HILIC that have emerged more recently, include the use of long

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columns to achieve highly efficient separations, a superior loading capacity to RP for charged

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basic solutes and the potential of very fast analysis due to the good mass transfer

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characteristics of the columns operated in mobile phases of much lower viscosity than

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typically used in RPLC [24, 26]. Nevertheless, HILIC also has some drawbacks [27]

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compared to RP separations which include: (i) the separation mechanism is at present less

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well understood than that of RPLC. Thus, it may be difficult to predict the effect of a change 6

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in conditions on the separation outcome, (ii) the technique does not have the broad

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applicability of RPLC. Analytes that are neutral and non-polar generally show very little

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retention. In addition, ionised acidic analytes (negatively charged ions) can show little

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retention due to repulsion of the ion from negatively charged column silanol groups on some

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silica-based columns and (iii) HILIC is potentially an environmentally less friendly technique

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than RPLC as it consumes much larger volumes of organic solvents.

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2. Stationary and mobile phases for HILIC applications

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2.1. Stationary phases in HILIC

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The chemical modification of silica gels using chemical reactions began in the 1960s.

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Reaction between alkylsilyl chlorides and silanol groups (Si–OH) is the most common

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method and was reported in 1970 [28]. Approximately at the same time as Alpert [9]

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introduced the HILIC technique, Huber et al. [29] noted that the correct selection of a suitable

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adsorbent is a crucial step for the success of so-called “solvent generated” liquid-liquid

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chromatography, the category to which the HILIC mechanism can be attributed [30]. The

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family of HILIC stationary phases with various support materials and surface chemistries has

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continuously enlarged since, to suit specific separation problems. The basic types of HILIC

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columns include plain silica, neutral, polar chemically bonded, ion-exchange and zwitterionic

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stationary phases. NP separations on bare silica and amino-silica columns in aqueous-organic

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mobile phases were reported as early as the mid-seventies [31]. Using neutral stationary

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phases (e.g. diol and amide phases) there will be no electrostatic interactions, whereas with

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charged phases (e.g. plain silica and aminopropyl phases) strong electrostatic interactions can

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take place between the analytes and the stationary phase. Zwitterionic phases (e.g.

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sulfobetaine silica) exhibit only weak electrostatic interactions with analytes [1]. The first separations in HILIC mode were reported in 1975. Linden et al. [31] separated

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carbohydrates using an amino-silica phase, Bondapak (Waters, Milford, MA, USA) in a

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mixture of ACN and water (75:25 v/v). The next generation of stationary phases for HILIC

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used diol- and amide-silica. The diol-silica column has mainly been used for the separation of

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proteins [32]. Amide-silica columns have been available since the mid-eighties. This

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particular phase is described as consisting of non-ionic carbamoyl groups that are chemically

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bonded to the silica gel, but is commonly known as an amide bonded silica. After Yoshida

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[33] applied these phases for the separation of peptides, the amide-silica phase soon found

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common usage in HILIC. Chemically bonded stationary phases with specific structural

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properties have been prepared by Buszewski et al. [34, 35]. Since then silica hydride, hybrid

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silica-organic phases and a large variety of silica-based or polymer HILIC columns have been

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designed. Amide-, cyano-, diol-, polyethylene glycol-, poly(succinimide)- , sulfoalkylbetaine-,

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cyclodextrin, pentafluorophenylpropyl, polyvinylalcohol, polypeptide and other polar

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chemically bonded stationary phases have become available for HILIC separations, some of

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them showing both HILIC and RP application possibilities [10]. Hence, the structural

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variations of HILIC-type stationary phases are wider than those found in reversed-phase

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systems. Table 1 shows a summary of the different structures of stationary phases that are

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applicable to HILIC-mode separation [11].

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Chirita et al. [36] suggested a good scheme for column selection and applied it to the

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analysis of neurotransmitters. In terms of the retention characteristics and selectivity, their 8

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column selection method seems to be acceptable but the separation efficiency must be

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considered too. The characterization of HILIC stationary phases has been reported, but there

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is no test scheme to describe the structure selectivity relationships for HILIC phases, in

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contradistinction with the well-accepted column tests for RP stationary phases [37–40].

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Kawachi et al. [41] suggested an inclusive test scheme of HILIC stationary phases using

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trimethylphenylammonium chloride, sodium p-toluenesulfonate, nucleosides, saccharides,

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and xanthines to describe the degree of hydrophilicity, the selectivity for hydrophilic and

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hydrophobic groups, positional selectivity and the configuration of hydrophilic groups, the

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anion and cation exchange properties, the local pH conditions on the stationary phases and the

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shape selectivity. In this test scheme, it was possible to divide the HILIC phases into several

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groups with varying selectivity. This can be helpful to select stationary phases in combination

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with the structural characteristics of the target analyte.

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2.2. Mobile phases in HILIC

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solvents with a small amount of water [9]. Although the retention mechanism in HILIC is not

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completely understood, it is supposed to be caused by partitioning. This mode is based on the

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differential distribution of the injected analyte molecules between the organic-rich mobile

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phase and a water-enriched layer adsorbed onto the hydrophilic stationary phase. The more

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hydrophilic the analyte, the more the partitioning equilibrium is shifted towards the

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immobilized water layer on the stationary phase, and thus, the more the analyte is retained [9,

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12].

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Usually, ACN is used as organic solvent, but any aprotic solvent that is miscible with

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water (e.g., tetrahydrofuran, THF) can be used. Alcohols can also be adopted, although a

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higher concentration is needed to achieve the same degree of retention of the analyte relative

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to an aprotic solvent–water combination [12]. HILIC on polar stationary phases employs

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organic-rich mobile phases, usually containing 5–40% water or a volatile buffer if used in

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combination with MS or charged aerosol detection. Mobile phase pH and ionic strength

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significantly affect the retention and separation selectivity of ionisable analytes in HILIC

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separations which can be detrimental for the robustness. Buffer salts and mobile phase

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additives such as ammonium acetate, ammonium formate, ammonium phosphate and

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trifluoroacetic acid are commonly used to improve peak shape and to control analyte polarity

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so as to effect differential changes in retention. However the positive impact of mobile phase

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pH and ionic strength on a separation also depends on the properties of the stationary phase

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[10, 11]. It is recommended to perform initial experiments at a relatively high concentration

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(e.g., 40%) of aqueous buffer in ACN, to ensure the elution of all sample compounds. Then

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the elution strength is adjusted by increasing the concentration of ACN, until acceptable

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sample retention is achieved. Alternatively, a scouting gradient of decreasing ACN

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concentration can be run. Gradients with high initial concentration of organic modifier and

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increasing concentrations of water were successfully applied for HILIC separations of various

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biological samples, especially with bare silica columns. The gradient usually starts from 95%

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ACN containing 5% aqueous ammonium acetate or ammonium formate buffer, with gradually

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decreasing ACN concentration. Running the gradient up to a high water concentration (up to

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90%) may help removing strongly retained sample interferences [10].

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In HILIC water acts as the strong solvent, hence the higher the organic content the greater

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the retention of the polar analytes will be [42]. For this reason, solvents typically used in

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HILIC contain 60–90% of organics and are thus suitable for LC/MS analysis using ESI,

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without significant changes in ionization efficiency during gradient elution [1, 42]. In mobile

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phases containing some water, the preferential adsorption causes formation of a water-rich

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adsorbed liquid multi-layer (Fig. 2). In aqueous-organic NP containing more than 0.5–1%

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water, the layer of adsorbed water is thick enough to induce liquid-liquid partition between

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the bulk mobile phase and the adsorbed aqueous liquid layer. However, sample adsorption on

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polar surface centers may significantly contribute to the retention [10]. The NPLC drawback

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of insolubility of hydrophobic compounds is largely solved in HILIC because of its mobile

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phase properties. It often provides sufficient retention of strongly polar compounds, for which

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it offers different selectivity compared to traditional RP chromatography [9, 43].

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The selection of the organic solvent in HILIC has a strong effect on the retention. The

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elution strength of organic solvents in the HILIC mode increases generally in the order of

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increasing solvent polarity and ability to participate in proton-donor/proton-acceptor

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interactions: methanol > ethanol > 2-propanol > THF > ACN [43]. ACN is strongly preferred

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as the organic compound because mobile phases containing other solvents often provide

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insufficient sample retention and broad or non-symmetrical peak shapes [10]. Another

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interesting organic solvent to be used in the mobile phase is THF, which is a strong hydrogen

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bond acceptor which may result in a different analyte elution order than ACN. Polar protic

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solvents, such as methanol, ethanol or isopropanol can act as both hydrogen bond donors and

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acceptors and can compete for active polar sites on the HILIC surface. Therefore analytes,

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whose retention is based on strong hydrogen bonding, are poorly retained. Approaches for

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method development in HILIC have been extensively discussed in a publication by Dejaegher

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et al. [44].

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3. Use of HILIC for analysis of antibiotics

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3.1. Aminoglycoside antibiotics

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3.1.1. Analysis of aminoglycosides

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Aminoglycosides (AGs) are very polar and lack chromophores (neomycin B is shown as

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an example in Fig. 3), which makes them difficult to separate using conventional RPLC with

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UV detection. Researchers have overcome this problem by employing derivatization agents

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and ion-pair reagents for detection [53-63]. However, derivatization steps make the analytical

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process more time consuming and often give problems with quantitation. HILIC combined

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with MS has been receiving great attention for AGs analysis as there is no need for

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derivatization or addition of ion-pairing reagents.

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3.1.2. HILIC in the analysis of aminoglycosides HILIC has been widely used to analyze aminoglycosides in different matrices (Table 2),

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including muscle and kidney [64, 65], plasma [66], serum [67, 68], manure [69], apples [70],

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milk [71], honey [72] and bulk samples [73]. Kumar et al. [72] reported a HILIC method for

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the analysis and investigation of the chromatographic behavior of several AGs (streptomycin,

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dihydrostreptomycin, spectinomycin, apramycin, neomycin, paramomycin, kanamycin A and

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gentamicins). Stationary phases with three different charge states, such as bare silica 12

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(negative), aminopropyl (positive), amide (neutral) and ZIC HILIC (zwitterionic), were

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chosen for the screening experiments. Mobile phase constituents (pH and ionic strength) and

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column temperature were also investigated. The zwitterionic HILIC provided the most

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satisfactory separation and sensitivity. The method was directly applicable for the analysis of

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AG residues in honey. Based on these research results, Kumar et al. [74] continued to study

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AG residues in kidney samples from food-producing animals and in honey samples by

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zwitterionic HILIC in combination with a triple quadrupole mass analyzer. The LOQ ranged

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from 2 to 125 µg/kg in honey and 25 to 264 µg/kg in kidney samples, which is well below the

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maximum residue limits (MRLs) established. Also in other studies zwitterionic HILIC

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stationary phases were found to perform well [64, 67, 68, 72, 74]. Six AGs (amikacin,

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gentamicin, kanamycin, neomycin, paromomycin and tobramycin) were simultaneously

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quantified by ZIC HILIC combined with ESI-MS/MS detection in human serum [67].

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Similarly, neomycin was quantitated by LC-MS/MS using HILIC in human serum [68].

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Analysis of neomycin, apramycin and kanamycin was possible by zwitterionic HILIC with

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MS detection and the same column was also successfully employed in a retention mechanism

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study (Fig. 4) [75].

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built up when equilibrating the stationary phase with the mobile phase. A polar analyte will

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experience hydrophilic partitioning between the water layer and the mobile phase. Besides, a

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charged analyte may have weak and/or strong electrostatic interactions (both attraction and

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repulsion) by the zwitterionic groups, which greatly contribute to the separation [10]. Since

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the AGs have strong electrostatic interaction with the zwitterionic stationary phase, it is

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necessary to use formic acid and a high buffer concentration in the mobile phase to achieve

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A crucial part in the separation mechanism is a water layer, which is

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good chromatographic performance.

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Multi-residue AG antibiotics in bovine and porcine muscle and kidney were quantitated

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using LC-MS/MS [64]. A ZIC-HILIC column was employed for the chromatographic

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separation. The method was found to be sensitive, robust and highly specific for the

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simultaneous quantification of the AGs with LOQs of 25 ng/g for gentamicin, 50 ng/g for

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spectinomycin, dihydrostreptomycin, kanamycin and apramycin; and 100 ng/g for

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streptomycin and neomycin, which are all well below the maximum residue limits set by the

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Codex Alimentarius Commission [64].

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Streptomycin (STR) and dihydrostreptomycin (DHSTR) are two of the most commonly

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used AG antibiotics in veterinary medicine. Gremilogianni et al. [71] compared HILIC with

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ion-pair chromatography (IPC) for the determination of STR and DHSTR residues in milk.

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Comparison of the validation parameters (sensitivity, precision and recovery) of these two

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methods revealed superiority for the HILIC method (Table 3). HILIC separation was

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performed using a silica based HILIC Fortis column. The sensitivity of the HILIC method

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was greater than that of the IPC method for both STR and DHSTR.

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A HILIC-MS/MS method for detection and quantification of spectinomycin and

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lincomycin from liquid manure and rainfall run-off was described by Peru et al. [69]. The

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chromatographic separation was performed on a silica-based Altima HP HILIC column. MS

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detection was carried out using an atmospheric pressure chemical ionization (APCI) interface

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in multiple reaction monitoring (MRM) positive ion mode. They found that the method was

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sensitive for the quantification of the antibiotics and that HILIC provided excellent retention 14

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and separation of spectinomycin and lincomycin from interfering matrix components without

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the need for ion-pairing reagents.

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An automated LC-MS/MS method using a CAPCELL PAK ST HILIC column for the

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simultaneous quantification of 15 AGs residues in muscle, liver (pigs, chicken and cattle),

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kidney (pigs and cattle), cow milk and hen eggs was reported elsewhere [65]. The 15 AGs

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consist of streptomycin (STREP), amikacin (AMIKA), hygromycin B (HYGRO),

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dihydrostreptomycin (DIHY), netilmicin (NETIL), kasugamycin (KASUG), kanamycin B

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(KANA), sisomicin (SISO), spectinomycin (SPECT), gentamicin C1 (GENT), apramycin

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(APRA), paromomycin (PAROM), tobramycin (TOBRA), ribostamycin (RIBOS) and

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neomycin B (NEOM). The detection capabilities in this method ranged from 10 to 50 µg/kg.

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The MRM LC-MS/MS chromatograms of the extractions of blank bovine liver spiked with

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these AGs are shown in Fig. 5. The report concluded that the LC-MS/MS method could be

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applied to trace analysis of multi-component AG contaminants in complex sample matrices.

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The HILIC method gave satisfactory retention and separation of the AGs residues.

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3.2.

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Beta-lactam antibiotics

Penicillins and cephalosporins have been thoroughly investigated in RP-HPLC systems

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coupled with different methods of detection such as UV detection [76, 77], MS [78, 79] or

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chemiluminescence detection [80]. Since these compounds are rather small polar molecules,

320

they could hardly be analyzed in RP-HPLC mode. Ion-pairing reagents, buffers and acids are

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often used as additives [81-83] to improve the retention and peak shape [84]. In addition,

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multidimensional [85] and capillary HPLC systems have been developed to analyze 15

Page 15 of 60

cephalosporins at lower concentrations in different sample matrices [86]. Some

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cephalosporins were used as model substances in mixtures with other drugs in HILIC mode,

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but their particular chromatographic retention was not explained [23, 87]. It is reported that

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HILIC was also used to analyze cephalosporin C [88, 89].

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The simple mobile phase of HILIC and its compatibility with MS opened a new door for

329

the analysis of cephalosporins. Liu et al. [88] developed a HILIC method to separate seven

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commonly used cephalosporins (cefotaxime sodium, cefpiramide, cefazolin sodium, cefepime

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hydrochloride, cefixime, ceftazidime and ceftriaxone sodium). Chromatograms for the 7

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cephalosporins on the Click β-CD column and Atlantis HILIC silica column are shown in Fig.

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6. The orthogonality between HILIC and RPLC for cephalosporins was also investigated. The

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authors further developed a successful HILIC method to analyze cefotaxime sodium and its

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degradation products. A HILIC-MS/MS method for the simultaneous determination of three

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polar, non-structurally related compounds – (1) a carbapenem antibiotic, imipenem (IMP), (2)

337

a renal dehydropeptidase inhibitor, cilastatin (CIL) and (3) an investigational β-lactamase

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inhibitor MK-4698 (BLI) – in biological fluids was described elsewhere [89].

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HILIC-ESI-MS/MS method for the multitarget quantitative analysis of the hydrophilic

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metabolites of penicillins and cephalosporins has also been described [90]. Jovanović et al.

341

[91] reported the chromatographic behaviour of mixture of β-lactam antibiotics (Cefotaxime,

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cefalexin, cefaclor, cefuroxime, cefuroxime axetil, ampicillin and amoxicillin) using

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HILIC-UV and their retention prediction models were designed by applying Box-Behnken

344

Design. Beta-lactam antibiotics analyzed using HILIC with different detection systems are

345

summarized in Table 4.

Ac ce p

te

d

M

an

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cr

328

A

16

Page 16 of 60

346

3.3. Tetracycline antibiotics Tetracycline antibiotics (TCs) are a large class of antibiotics that characteristically contain

348

an octahydronaphthacene ring skeleton with four fused rings. The various TCs mainly differ

349

in their substitution groups at the C5, C6 and C7 positions and include tetracycline (TC),

350

chlortetracycline (CTC), doxycycline (DC) and oxytetracycline (OTC) (Fig.7).

cr

ip t

347

351

Analysis methods of TCs over the past twenty years have been reviewed [92, 93-98].

353

Among them, LC using a C18 or C8 column, with UV, fluorometry and mass spectrometry

354

detection are generally preferred. However, owing to the presence of two ketone groups in

355

positions 1 and 11, and the enols around C10 and C12, TCs can readily chelate with trace

356

metal ions present in silica packing material or from the equipment. Most of them have a

357

strong tendency to irreversibly bind to the silanol groups in a silica-based packing column. As

358

a result, this causes severe tailing of TCs peaks [99-102]. In order to avoid this problem,

359

adding chelating agents, such as oxalic acid and EDTA salts to the mobile phase and RP

360

end-capped columns have been employed. Using HILIC silica with high purity silica columns,

361

non-selective reactions such as complex formation or ion effects leading to less favorable

362

separations could be prevented [103, 104].

364

an

M

d

te

Ac ce p

363

us

352

TCs are not only widely used as veterinary medicines, but also as growth additives in

365

animal feeds or water [92]. It has been reported that only a small portion of the applied TCs

366

(veterinary or food additives) is metabolized or absorbed in animals and most of the

367

unmetabolized form is released in excreta [105], which enters into the environment and

368

disrupts the indigenous microbial population. Therefore, many researchers focus on the 17

Page 17 of 60

development of a rapid and sensitive method for the determination of TCs in the environment.

370

Li et al. [106] reported the environmental fate and transport of four zwitterionic TCs

371

(oxytetracycline, doxycycline, chlortetracycline and tetracycline) and developed a

372

HILIC-MS/MS method for detection and quantification of these antibiotics in environmental

373

waters. The chromatographic separation was achieved on an amino-bonded silica HILIC

374

column under isocratic conditions. During method development, the effects of mobile phase

375

components (buffer type, pH and type and concentration of organic modifier) on retention and

376

separation of TCs on the column was investigated and a mixed-mode retention mechanism

377

composed of partitioning, adsorption and ion-exchange interaction was proposed.

an

us

cr

ip t

369

M

378

Determination of OTC, TC and CTC in different sample matrices using a HILIC or mixed

380

HILIC-ion exchange mode were also described elsewhere [107, 108]. The methods gave rapid

381

and robust separations of the TCs. HILIC using high-purity silica as stationary phase has been

382

applied for the separation of epirubicin and its analogues [109]. Several parameters affecting

383

the chromatographic behavior such as organic modifier, buffer pH and ion strength have been

384

investigated. Of utmost importance for successful separation of the analogues (doxorubicin,

385

daunorubicin and epidaunorubicin) is the choice of organic modifier. ACN was shown to offer

386

superior separation to methanol, isopropanol or THF. Table 5 summarizes the application of

387

HILIC for TCs in different sample matrices.

Ac ce p

te

d

379

388 389

3.4. Other antibiotics

390

Besides β-lactams, AGs and TCs, HILIC has been widely employed for the analysis of

391

other antibiotics (Table 6). Although RPLC is the predominant LC mode applied to peptide 18

Page 18 of 60

separation, there is a growing awareness of the potential of HILIC as a complementary mode

393

in situations where RPLC does not offer the required retention and overall separation

394

efficiency. Determination of the glycopeptide antibiotic avoparcin in kidney using a 200Å, 5

395

µm HILIC column was reported by Curren and King [110]. Van Dorpe et al. [111] reported

396

HILIC analysis of peptide antibiotics (vancomycin and polymyxin) using Alltima HP and ZIC

397

HILIC columns. Determination of 13 sulfonamide antibacterials in milk and eggs using the

398

polymer monolith microextraction (PMME) technique was also possible [112] on a Luna NH2

399

HILIC column followed by MS detection.

400

carbadox and olaquindox with matrix solid-phase dispersion (MSPD) extraction in swine feed

401

using a BEH HILIC column [113] and bicozamycin using a TSK-GEL NH2 HILIC column

402

and MS detection [114] showed to be successful with greater separation efficiency. Analysis

403

of levofloxacin in human plasma using an Atlantis HILIC silica column and determination of

404

gemifloxacin on dried blood samples using

405

reported [115, 116] and the methods were successfully applied for pharmacokinetic studies of

406

these drugs.

408 409

us

cr

te

d

M

an

Quantification of antibacterial drugs like

a ZIC-HILIC C18 column have also been

Ac ce p

407

ip t

392

4. Conclusion

The use of HILIC for the analysis of antibiotics is reviewed. In recent years, the HILIC

410

separations have received greater attention mainly due to their versatility for the analysis of

411

polar drugs, metabolites, biologically important compounds, etc. in complex sample matrices

412

of different origin. Due to the very hydrophilic nature of most antibiotics, HILIC is more

413

suitable for the analysis of antibiotics compared to traditional RP chromatography. There are 19

Page 19 of 60

414

many kinds of HILIC columns available on the market with diverse stationary phases.

415

However, bare silica, zwitterionic and aminopropyl based stationary phases are most

416

commonly used.

417

separation of most antibiotics. The on-going developments of new HILIC methods in

418

conjunction with more powerful detection systems will probably increase its use and

419

popularity in antibiotics analysis in the future.

us

cr

ip t

ESI-MS/MS is the most popular detection method employed in HILIC

420

an

421 422

M

423

427 428 429 430 431

te

426

Ac ce p

425

d

424

432 433 434 435 20

Page 20 of 60

Figure legends

437

Fig. 1. HILIC as a complement to normal phase and reversed phase LC separations.

438

Fig. 2. Schematic picture of an adsorbed diffuse water layer at the surface of a polar stationary

439

phase in highly organic environment. Figure adapted from [10].

440

Fig. 3. Chemical structure of neomycin B

441

Fig. 4. Retention of polar/hydrophilic compounds on a zwitterionic HILIC stationary phase.

cr

Figure adapted from [75].

us

442

ip t

436

Fig. 5. MRM LC-MS/MS chromatograms of the extractions of blank bovine liver spiked with

444

AGs equivalent to detection capabilities of 20 µg/kg for APRA, STREP, DIHY, SPECT,

445

GENT, TOBRA, PAROM, HYGRO, RIBOS, KASUG, AMIKA, NEOM, KANA, SISO,

446

NETIL. Figure adapted from [65].

d

447

M

an

443

Fig. 6. Chromatograms for seven cephalosporins on the Click β-CD column (a) and Atlantis

449

HILIC Silica column (b, c). Mobile phase: A, 10 mM ammonium formate at pH 6.8; B,

450

ACN-100 mM ammonium formate = 90:10 at pH 6.8. Gradient for (a) and (b) was

451

88%–65%B in 20 min and 65%B in the next 10 min; gradient for (c) was 100%–75%B in 20

452

min and 75%B in the next 15 min. (1) Cefotaxime sodium, (2) cefpiramide, (3) cefazolin

453

sodium, (4) cefepime hydrochloride, (5) cefixime, (6) ceftazidime and

454

sodium. Figure adapted from [88].

Ac ce p

te

448

(7) ceftriaxone

455 456

Fig. 7. Structure of some tetracyclines

457 458 21

Page 21 of 60

459 460 461

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711

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713

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761

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846 847 848

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851

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interaction 38

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ultra-high-pressure liquid chromatographic analysis, J. Chromatogr. A 1209 (2008)

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83–87. [114] K. Inoue, E. Yamada, T. Hino, H. Oka, Hydrophilic Interaction Liquid

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Chromatography Tandem Mass Spectrometry Method for the Determination of

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ip t

858

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levofloxacin in human plasma, J. Pharm. Biomed. Anal. 41 (2006) 622–627.

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M

865

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mass spectrometry, J. Chromatogr. A 1217 (2010) 3076–3084.

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hydrophilic interaction liquid chromatography-tandem mass spectrometry for the

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877

117–129.

39

Page 39 of 60

*Graphical Abstract

cr

Ion Chromatography

Normal Phase

ip t

Graphical Abstract

us

HILIC

Ac

ce pt

ed

M

an

Reversed Phase

Page 40 of 60

*Highlights (for review)

Highlights ► Use of HILIC for the analysis of antibiotics is reviewed. ► Specific applications of HILIC for different classes of antibiotics are discussed.

ip t

► ESI-MS/MS is the most popular detection method in HILIC separation of most antibiotics.

Ac

ce pt

ed

M

an

us

cr

► It provides comprehensive insight into HILIC application to antibiotics analysis.

Page 41 of 60

ip t

Table(s)

Packing materials

cr

Table 1 Summary of stationary phases used in HILIC separations [11]. Structure of stationary phase Siloxane

Silanol OH HO

siloxanes, silanols with (or without) a

Si

O

O

Si

OH

O

[12, 23]

Si

O

OH Si

derivatives

Diol bonded phases

Ac c

Amino bonded phases

ep te

Polymeric structures of poly (succinimide)

d

M

small quantity of metals

Si

OH

an

contain functional groups such as

us

Underivatized silica stationary phases that

Application

[23]

[23, 31]

[32]

Page 42 of 60

ip t cr an

us

Amide bonded phases

[34]

Mix-mode

Polyethylene

Ac c

Cyano bonded phases

ep te

d

M

Alkylamide

[23, 33]

glycol/silica (HS PEG)

[35]

[45]

[46]

Page 43 of 60

ip t cr

[47]

an

us

“click” ß-cyclodextrin

M

“click” saccharides

[48]

ep te

d

(“click” maltose)

Ac c

“click” dipeptide

[49]

Zwitterionic sulfobetaine

bonded phases (ZIC-HILIC)

[50]

Page 44 of 60

ip t cr

Cationic exchangers

us

bonded phases

an

Mix-mode RP/anionic exchangers bonded

[50, 52]

Ac c

ep te

d

M

phases

[51]

Page 45 of 60

ip t cr

Table 2 HILIC applications in the analysis of aminoglycosides Compound (s) of interest

Stationary phase

Kidney and muscle

Gentamicin/spectinomycin/dihydrostrept

tissues

omycin/kanamycin/apramycin/streptomy

Detection

Refs

ZIC-HILIC (SeQuant)

150 mM ammonium acetate +

ESI-MS

[64]

(100 mm x 2.1 mm, 5 µm)

1% FA-ACN; gradient

ACN-0.1% TFA; gradient

ESI-MS/MS

[65]

ESI-MS

[66]

ESI-MS/MS

[67]

an

M

cin/neomycin

Mobile phase

us

Matrix

Streptomycin/ amikacin/hygromycin

kidney, cow milk

B/dihydrostreptomycin/netilmicin/

and hen eggs

kasugamycin/ kanamycin B/ sisomicin/

CAPCELL PAK ST (150 mm x 2.0 mm, 3 µm)

ep te

d

Muscle, liver,

spectinomycin/ gentamicin

C1/apramycin/ paromomycin/

Ac c

tobramycin/ ribostamycin/neomycin B Mouse, Rat and

Amikacin/streptomycin/spectinomycin/ge ZIC-HILIC (SeQuant)

5 or 25 mM ammonium

Guinea Pig plasma

ntamicin

formate, pH 2.5-ACN - 1%

Human serum

(50 mm x 2.0 mm, 5 µm)

FA ; gradient

Amikacin/gentamicin/kanamycin/neomyc ZIC-HILIC

ACN-2 mM ammonium

in/paromomycin/ tobramycin

acetate- FA; gradient

(100 mm x 2.1 mm)

Page 46 of 60

ZIC-HILIC

ip t

Neomycin

[68]

APCI-MS/MS

[69]

ESI-MS/MS

[70]

ESI-MS

[71]

ESI-MS/MS

[72]

[73]

acetate- FA; gradient

(150 mm x 2.1 mm, 3 µm)

isocratic

Atlantis HILIC Silica

A: water + 0.05% FA /B:

(150 mm x 2.1 mm, 3 µm)

ACN + 0.05% FA; gradient

HILIC Fortis

150 mM ammonium formate-

(100 mm x 3.0 mm, 3 µm)

ACN, pH 4.5; gradient

Streptomycin/spectinomycin/dihydrostre

ZIC-HILIC

175 mM Ammonium formate ,

ptomycin/gentamicin C1/ gentamicin

(150 mm x 2.1 mm, 3.5

pH 4.5- 0.2% FA in ACN;

C1a/gentamicin C2/C2a/

µm)

gradient

Impurities in streptomycin and

Halo HILIC

100–200 mM ammonium

ESI-

dihydrostreptomycin

(100 mm x 2.1 mm, 2.7

formate, pH: 3.0–4.5-ACN;

QIT/TOFMS

µm)

isocratic

an

ACN-water-0.1% FA;

Streptomycin

Streptomycin/dihydrostreptomycin

ep te

Honey

ESI-MS/MS

Altima HP HILIC

d

Milk

Spectinomycin/lincomycin

M

Apples

us

(100 mm x 2.1 mm ) Manure

ACN-10 mM ammonium

cr

Human serum

Ac c

Apramycin/paromomycin/kanamycin A /neomycin B Bulk samples

FA : Formic acid; TFA: Trifluroacetic acid; QIT: Quadrupole ion trap

Page 47 of 60

Table 3 Comparison of the validation parameters of HILIC versus IPC method [71]. IPC method

STR

DHSTR

STR

DHSTR

LOQ

13.9 µg/kg

14 µg/kg

109 µg/kg

31 µg/kg

Recovery

85.5%

72.3%

69.3%

Inter-day precision

6.7–8.1%

9.8–11.2%

8.9–12.1% 9.7–13.4%

ip t

HILIC method

Ac

ce pt

ed

M

an

us

cr

56.5%

7

Page 48 of 60

ip t

Compound (s) of interest

Stationary phase

Mobile phase

Detection

Refs

Fermentation

2-Aminoadipic acid , 2-Amino-5-(4-carboxy-2-

ZIC-HILIC (and ZIC-

A: 10 % (v/v) 200

ESI-MS/MS

[88]

broths

thiazolyl)-valeric acid, 6-Aminopenicillanic acid,

HILIC Guard 20 mm x 2.1

mM FA, pH 4.0 in

6-Aminopenicilloic acid, 7-Aminocephalosporanic

mm; 5 µm)

water /B: 10 % (v/v)

A: 10 mM ammonium

UV/Vis ESI-

[89]

MS/MS

an

cr

Matrix

us

Table 4 Summary of beta-lactam antibiotics analyzed using HILIC with different detection systems

200 mM FA, pH 4.0

Cephalosporin C lactone, Deacetoxycephalosporin,

in ACN; gradient

M

acid, 8-Hydroxypenillic acid, Cephalosporin C,

Deacetylcephalosporin, Penicillamine disulfide,

d

Phenoxyacetic acid, Phenoxymethylpenicilloic

ep te

acid, Phenoxymethylpenillic acid, pHydroxyphenoxyacetic acid, δ-(L-αAminoadipoyl)-L-Cys-D-Val

Ceftazidime, cefixime, cefpiramide, ceftriaxone Atlantis HILIC silica

materials

sodium, cefotaxime sodium, cefazolin sodium, (100 mm x 2.1 mm, 5 µm)

formate, pH 6.8/B:

cefepime hydrochloride and degradation products Click β-CD column

ACN-100 mM

of cefotaxime sodium

ammonium formate,

Ac c

Raw

(150 mm x 2.1 mm, 5µm)

pH 6.8; gradient

8

Page 49 of 60

ip t

(50 mm x 2.1 mm, 3 µm)

Cefotaxime,

us

cefaclor,

cefuroxime, Alltech Silica

(250 mm x 4.6 mm, 5 µm)

[90]

UV/Vis

[91]

formate, pH 3 in 80% ACN; isocratic ACN-ammonium acetate, pH 4.5–6.5; isocratic

d

M

cefuroxime axetil, ampicillin and amoxicillin

an

cefalexin,

TIS-MS/MS

ep te

substances

lactamase inhibitor (BLI)

15 mM ammonium

Ac c

Reference

cr

Plasma, blood Imipenem (IMP), cilastatin (CIL), MK-4698 beta- Atlantis HILIC silica

9

Page 50 of 60

ip t Compound (s) of

Stationary phase

Detection Refs

ACN-6.7 mM ammonium citrate, pH

UV-Vis

[106]

UV-Vis

[107]

UV-Vis

[108]

UV-Vis

[109]

Mobile phase

us

Matrix

cr

Table 5 HILIC in the analysis of tetracylines antibiotics

interest OTC, TC, CTC,

Kromasil 100-5 NH2

water

doxycycline

(250 mm x 4.6 mm, 5 µm) 4.0 (85:15, v/v); isocratic

Environmental

OTC

Kromasil KR100-5SIL

M

ACN-10 mM oxalate, pH 2.5 (90:10,

(250 mm x 4.6 mm, 5 µm) v/v); isocratic ThermoHypersil APS2

ep te

TC, CTC, OTC

d

waters Drug substances

an

Environmental

ACN-6.7 mM citrate, pH 5.0 (85:15,

(50 mm x 4.6 mm, 3 µm)

v/v); isocratic

Epirubicin raw

Epidaunorubicin,

Kromasil KR100-5SIL

30 mM sodium formate, pH 2.9-ACN,

material

daunorubicin, epirubicin,

(250 mm x 4.6mm, 5 µm)

(90:10, v/v); isocratic

Ac c

doxorubicin

10

Page 51 of 60

ip t Compounds of interest

Stationary phase

Mobile phase

Detection

Refs

Kidney

Avoparcin

polyhydroxyethyl aspartamide

47% 15 mM Triethyl ammonium

UV

[110]

HILIC

phosphate in ACN; isocratic

[111]

an

us

Matrix

cr

Table 6 HILIC in the analysis of other antibiotics

(200 mm x 4.6 mm, 5 µm)

Sulfonamides antibacterial residues

Alltima HP HILIC

A: 0.1% w/v FA in ACN

Photodiode

(250 mm x 2.1 mm, 5 µm)

B: 0.1% w/v FA in water

array detector

Luna HILIC

ACN + 0.05% FA-water; gradient

ESI-MS

[112]

BEH HILIC

10 mM ammonium acetate in

Photodiode

[113]

(100 mm x 2.1 mm, 1.7 µm)

ACN -water (95:5, v/v); isocratic

array detector

TSK-Gel Amide 80

50 mM ammonium acetate in

ESI-MS/MS

[114]

(150 mm x 2.0 mm, 5 µm)

water (pH 4)-ACN; gradient

Atlantis HILIC Silica

ACN-100 mM ammonium

MS/MS

[115]

(50 mm x 3 mm, 5 µm )

formate , pH 6.5 (82:18 v/v);

Fluorescence

[116]

ESI-MS

[117]

M

Milk and eggs

Vancomycin and Polymyxin

d

Bulk substances

(150 mm x 2.0 mm, 3 µm)

Human plasma

Rat blood

Distillers grains

Bicozamycin

Levofloxacin

ep te

Milk

Carbadox and olaquindox

Ac c

Swine feed

Gemifloxacin

gradient ZIC-HILIC-C18

ACN -10 mM ammonium acetate

(100 mm x 4.6 mm, 5 µm)

(pH 3.5, 80:20, v/v); isocratic

Ampicillin, Penicillin G, OTC, TC,

Atlantic HILIC Silica

0.1% aqueous FA- ACN; gradient

CTC, Bacitracin A, Virginiamycin

(100 mm x 2.1 mm, 5 µm)

11

Page 52 of 60

ip t cr

Clarithromycin, Monensin A, STR ZIC–HILIC

β-lactams (3), lincosamides (2),

(2.1 mm x 100 mm, 3.5 µm)

macrolides (4), quinolones (4), sulfonamides (4) and tetracyclines

ESI-MS/MS

[118]

pH 2.5; B: ACN; gradient

ep te

d

M

(3)

A: 50 mM ammonium formate,

an

Aminoglycosides (3), amprolium,

Ac c

Chicken muscle

us

M1, Erythromycin A, Tylosin A,

12

Page 53 of 60

Figure(s)

ip t

High sensitivity

cr

HILIC

ESI-MS sensitivity

an

us

Reversed Phase

M

Normal Phase

Analyte Polarity

Apolar

Ac

ce pt

Polar

ed

Low sensitivity

Fig. 1

1 Page 54 of 60

Fig. 2

2

Page 55 of 60

ed

ce pt

Ac

us

an

M

ip t

Aqueous sublayer

cr

Polar stationary phase

Mostly organic mobile phase

ip t cr us an M ed ce pt Ac

Fig. 3

3 Page 56 of 60

ip t cr us an M ed ce pt Ac

Fig. 4

4 Page 57 of 60

ip t cr us an M ed ce pt Ac

Fig. 5

5 Page 58 of 60

ip t cr us an M d ep te Ac c

Fig. 6

6 Page 59 of 60

ip t R4

Teteracycline (TC)

H

OH

CH3

H

Chlortetracyclin (CTC)

H

OH

CH3

Cl

Oxytetracycline (OTC)

OH

OH

CH3

H

Doxycycline (DC)

H

H

CH3

cr

R3

us

R2

an

R1

Ac

ce pt

ed

M

H

Fig. 7

7 Page 60 of 60