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Evidence Project Final Report

NoteIn line with the Freedom of Information Act 2000, Defra aims to place the results of its completed research projects in the public domain wherever possible. The Evidence Project Final Report is designed to capture the information on the results and outputs of Defra-funded research in a format that is easily publishable through the Defra websiteAn Evidence Project Final Report must be completed for all projects.

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Project identification

1. Defra Project code VM02175

2. Project title

Further investigations into the use of time of flight mass spectrometry for rapid screening of veterinary medicine residues in animal tissues

3. Contractororganisation(s)

Food and Environment Research Agency(Formerly Central Science Laboratory)Sand HuttonYorkNorth YorkshireYO41 1LZ

54. Total Defra project costs £ 89938(agreed fixed price)

5. Project: start date 01/04/2010

EVID4 Evidence Project Final Report (Rev. 06/11) of 18

mailto:[email protected]

end date 31/03/21012


6. It is Defra’s intention to publish this form. Please confirm your agreement to do so...................................................................................YES NO (a) When preparing Evidence Project Final Reports contractors should bear in mind that Defra intends that

they be made public. They should be written in a clear and concise manner and represent a full account of the research project which someone not closely associated with the project can follow.Defra recognises that in a small minority of cases there may be information, such as intellectual property or commercially confidential data, used in or generated by the research project, which should not be disclosed. In these cases, such information should be detailed in a separate annex (not to be published) so that the Evidence Project Final Report can be placed in the public domain. Where it is impossible to complete the Final Report without including references to any sensitive or confidential data, the information should be included and section (b) completed. NB: only in exceptional circumstances will Defra expect contractors to give a "No" answer.In all cases, reasons for withholding information must be fully in line with exemptions under the Environmental Information Regulations or the Freedom of Information Act 2000.

(b) If you have answered NO, please explain why the Final report should not be released into public domain

Executive Summary7. The executive summary must not exceed 2 sides in total of A4 and should be understandable to the intelligent

non-scientist. It should cover the main objectives, methods and findings of the research, together with any other significant events and options for new work.


BackgroundThe Food and Environment Research Agency (FERA), had previously been commissioned to undertake an investigation into the potential use of Liquid Chromatography coupled to Time-of-Flight Mass Spectrometry (LC-ToF-MS) for the multi-class, multi-residue screening of samples for residues of veterinary medicines and related compounds (project VM02152). This resulted in the development of a broadly applicable multi-class, multi-analyte screening method. Because of the range of properties of analytes, some compromises had to be made in the development, which rendered the procedure unsuitable for some compounds, particularly at the polar end of the spectrum. One of the key recommendations coming out of this project was to investigate alternative conditions for the most polar compounds. These included the aminoglycosides, but also other compounds such as florfenicol amine, the sulfonamides sulfanilamide and sulfaguanidine and the insecticides cyromazine and dicyclanil.

The detection, identification and quantification of veterinary medicines and their metabolites and/or marker residues of use is time-consuming and costly as a number of class-specific methods, such as Liquid Chromatography – coupled to tandem Mass Spectrometry (LC-MS/MS) or bioassays, are required to provide a comprehensive screen. Bioassays suffer from the disadvantage of providing only limited information as to the identity of the analyte eliciting a response in a suspect sample. Consequently, and at increased cost, multiple confirmatory tests based on LC-MS/MS are often carried out in an attempt to By contrast, Time-of-Flight Mass Spectrometry (ToF-MS) captures high mass accuracy (mDa) mass spectra continuously across the entire mass range and chromatogram without compromising sensitivity. The aim of this project was to extend the number of compounds that could be detected using a single extraction method by inclusion of a number that had proved problematic within the previously developed protocol.

Main Project ObjectivesThe main project objectives are summarised as follows:

1. To develop alternative LC-ToF-MS conditions for those compounds of interest.2. To optimise the extraction and clean-up method for those compounds of interest3. To validate the optimised method.4. To evaluate the effect of any changes on analytes successfully validated under VM021525. To pass on the developed method to laboratories carrying out surveillance work.

Results and Key FindingsFour alternative LC-ToF-MS procedures were developed. The first requires the final extract to be dissolved in water rather than 1:1 methanol:water. As a result, more polar compounds focus better on the LC column, meaning that peaks are sharper and more reliably detected. The remaining three techniques result in polar compounds being more highly retained, with better peak shape and moving them away from matrix co-extractives that would otherwise suppress their ionisation, preventing their detection. Hydrophilic interaction chromatography (HILIC) is an alternative to the more commonly used reversed phase mode of LC separations. The use of ion-pairing reagents in the dissolution solvent is applicable to those compounds that are ionised or ionisable. In this instance, since most of the analytes of interest have the potential to form cations, an anionic ion-paring reagent (heptafluorobutyric acid) was used. The fourth procedure involved the use of a deriviatising agent (phenyl isocyanate). This works by reacting with primary or secondary amines to form urea derivatives. This can dramatically change the retention properties of the analyte and also the best ionisation mode for the ToF-MS analysis, but is not applicable to all the compounds of interest.

Each of these four protocols may be employed with minimal alterations to the generic protocol previously developed, by taking one aliquot and treating as previously and taking other aliquots to be treated as indicated above. Optiimisation experiments demonstrated that no one procedure was appropriate for all compounds of interest. In particular, the aminoglycosides as a class were not detected. Further experiments indicated that an alternative extraction solvent was required to successfully recover and detect the aminoglycosides. A two-stage extraction protocol was developed and proof-of-principle demonstrated. However the increase in complexity of the protocol meant that no further work was undertaken.Procedures based on water dissolution and HILIC chromatography were fully validated (CCß data) in bovine muscle. In addition, single validation batches were carried out in bovine muscle using the phenyl isocyanate/base derivatisation procedure. Data was provided using both pyridine and triethylamine as base. In addition single validation batches in bovine kidney and liver were carried out using the water dissolution and HILIC procedures. Single validation batches in salmon muscle were also carried out using


all three protocols.The structure of the developed protocol means that the data gathered under the previous project remains valid. Additional evidence was provided by the recovery of a range of analytes in an aliquot processed using the original procedure. Method transfer has been facilitated by preparation of a draft SOP and made available to the surveillance team.

Project Report to Defra8. As a guide this report should be no longer than 20 sides of A4. This report is to provide Defra with details of

the outputs of the research project for internal purposes; to meet the terms of the contract; and to allow Defra to publish details of the outputs to meet Environmental Information Regulation or Freedom of Information obligations. This short report to Defra does not preclude contractors from also seeking to publish a full, formal scientific report/paper in an appropriate scientific or other journal/publication. Indeed, Defra actively encourages such publications as part of the contract terms. The report to Defra should include: the objectives as set out in the contract; the extent to which the objectives set out in the contract have been met; details of methods used and the results obtained, including statistical analysis (if appropriate); a discussion of the results and their reliability; the main implications of the findings; possible future work; and any action resulting from the research (e.g. IP, Knowledge Exchange).

Project Objectives1. Determine optimum LC-ToF-MS conditions for compounds of interest.

2. Optimise extraction and clean-up method for compounds of interest.

3. Validation of the developed method.

4. Evaluate broader applicability of method to remaining analytes in previously developed screening procedure.

5. Transfer methods to surveillance laboratories.

1. Objective 1. Determine optimum LC-ToF-MS conditions for compounds of interest.

60 compounds were chosen for the initial investigation. Most were chosen because of their higher polarity, which resulted in poor chromatography and retention using the generic column and conditions developed previously in project VM02152. The remainder were chosen because of high levels of suppression and mass drift under the generic conditions, possibly linked to the presence of matrix co-extractives. Of particular interest were the aminoglycoside antibiotics (difficult analytes to retain and separate without the inclusion of ion-pairing reagents in the LC mobile phases) and florfenicol amine (for which methodology was required to service another project, VM02163).Four potential alternative systems were investigated: 1) HILIC using a variety of phases; 2) isocyanate derivatisation of the amine functional group common to a number of compounds of interest; 3) solvent switching of the redissolution solvent to water to enable improved focusing of the analytes on the analytical column under reversed phase conditions, yielding better peak shape and signal-to-noise and in some instances improved retention; 4) inclusion of ion-pairing reagent in the dissolution solvent only.

HILIC ChromatographyHydrophilic Interaction Liquid Chromatography (HILIC) is commonly perceived as a homogenous separation technique offering an alternative selectivity to standard reversed phase liquid chromatography (RPLC). However, there are numerous phases offering different functionalities. These range from bare silica surfaces to embedded zwitterionic phases. As with reversed phase columns, this means that secondary interactions may become important in separations. Table 1 summarises the phases investigated and their functionalities.The Hypercarb phase is not usually regarded as a HILIC phase, customarily showing properties more akin


to reversed or normal phase separations, depending on conditions. It did, however, show unanticipated retention properties for some compounds when used under HILIC conditions and was in consequence included in the investigation. A variety of mobile phase conditions, varying the pH and buffer type and concentrations, were investigated.

Table 1. Summary of HILIC phases investigated.

It was found, as had previously been demonstrated in the open literature [1], that whilst many of the compounds of interest chromatograph successfully at low buffer concentrations, high buffer concentrations (high ionic strength) buffers are required for the aminoglycoside and aminocyclitols antibiotics. Unfortunately, the high buffer concentration has a detrimental effect on sensitivities in general, due to suppression effects. It was concluded that there was still sufficient sensitivity to detect at least some of the compounds of interest at the target concentrations. After detailed study, two columns, the BEH HILIC and zic-HILIC, and two sets of conditions were selected as determinative procedures for the extraction and clean-up investigation. In both sets of conditions the organic mobile phase was acetonitrile. The first set of conditions used an aqueous mobile phase of 1% formic acid in 0.15M ammonium acetate, with two gradient steps separated by an isocratic step (modified from [1]). The second set used an aqueous mobile phase of 0.05M ammonium formate buffered to pH2.5 with formic acid, with two gradient steps (modified from [2]). Although the buffer concentration in this second set of conditions appears to be much lower than in the first set, the volume of formic acid used to reduce the pH to 2.5 is such that the ionic strength of the aqueous component is sufficiently high for the successful chromatography of the aminoglycosides.

The alternative selectivity of HILIC to RPLC is illustrated in figure 1 for amprolium, a coccidiostat that is a quaternary ammonium salt.

Figure 1. Improved retention and peak shape under HILIC conditions – Amprolium as an example.


HILIC(BEH HILIC)

Reversed Phase(HSS T3)

AmproliumN

N

N+

NH2

Cl

1.2.. Isocyanate derivatisationThe use of phenyl isocyanate to derivatise aminoglycosides prior to determination by LC-MS/MS has been reported in the open literature [3]. The isocyanate reacts with primary and secondary amines in the presence of base to form a urea derivative (see figure 2). This has the effect of reducing the polarity of the derivative compared to the underivatised compound, resulting in increased retention under reversed phase conditions and changing the optimum mode of detection from positive to negative. Since aminoglycosides have multiple amino groups, they have the potential to undergo multiple reactions. The effect on retention is dramatic.

Figure 2. reaction of isocyanates with primary and secondary amines.

A number of the compounds on the target list other than aminoglycosides also contain primary or secondary amino groups and would be expected to react in a similar manner with isocyanate reagents. These include florfenicol amine and the sulfonamides, for example sulfaguanidine and sulfanilamide, both difficult to detect using conventional chromatographic separations. It was thought that it might be possible to improve the derivatisation reaction and also change the optimum mode of ionisation of the derivatives by changing the structure of the aromatic group on the isocyanate. In order to investigate these possibilities, two compounds were chosen as suitable test analytes, neomycin B (6 amino groups and the potential to form multiple derivatives) and florfenicol amine (which can only form a mono derivative). Four isocyanates (phenyl, 4-dimethylamino, 4-nitrophenyl, 2,4-dichlorophenyl) were investigated along with two bases, pyridine and triethylamine. The results from these experiments are summarised in Table 2.

Table 2. Relative responses of four isocyanate derivatives, normalised against phenyl isocyanate derivative in positive mode.

PhIC - phenyl isocyanate, DMAPhIC - 4-dimethylaminophenyl isocyanate, NPhIC - 4-nitrophenyl isocyanate, DCPhIC - 2,4-dichlorophenyl isocyanate

From these data, it can be seen that phenyl, 4-nitrophenyl and 2,4-dichlorophenyl isocyanate derivatives can be better detected in negative ionisation mode whereas derivatives of 4-dimethylaminophenyl isocyanate respond better in negative ionisation mode. Phenyl isocyanate/pyridine/negative ionisation mode are the conditions of choice for neomycin B whereas 2-dimethylaminophenyl


isocyanate/pyridine/positive ionisation mode are better conditions for florfenicol amine. These latter conditions would be those of choice for a single residiue method, but use of phenyl isocyanate appeared to give a broader coverage of analytes. Although pyridine appeared to give marginally better results with this reagent, both bases were carried forward for further investigation. The effect of derivatisation on retention and chromatography is illustrated in figure 3 using florfenicol amine as a model.

Figure 3. Comparision of retention under reversed phase conditions for derivatised and underivatised florfenicol amine.

1.3. Water as dissolution/injection solventProbably the simplest approach to modifying the generic method previously developed to include the more polar analytes is to modify the final dissolution solvent. This section and the following deal with two different ways of doing this.1:1 methanol:water was chosen in the original method development (VM02152) as a compromise because of the broad range of polarities of the compounds of interest. Water was not appropriate as a dissolution solvent for the more non-polar compounds due to losses at either the dissolution or the filtration step. Using water as injection solvent is unlikely to cause major shifts in retention time (cf sections 1.1, 1.2) it does offer the advantage of allowing the more polar analytes to focus better at the top of the analytical column, so that improved peak shape and sensitivity is achieved. The co-extractives in the final extract may also differ.The technique was found to improve peak shape and sensitivity for a number of the target compounds (for


Derivatised(Phenyl isocyanate)

Underivatised

Florfenicol amine

OH

F

NH2

SOO

example the ectoparasiticide cyromazine, see figure 4). However there is little advantage to be gained for those compounds such as the aminoglycosides that chromatograph at or close to the solvent front. Some degree of retention is required for this technique to offer an advantage.

Figure 4. Use of water as dissolution/injection solvent - cyromazine as example.1.4. Use of Ion-Pairing ReagentsAs indicated above, changing the final dissolution solvent to water does not offer any real advantage to the detection of those compounds, such as the aminoglycosides, which run at or close to the solvent front under standard reversed phase conditions. Ion-pairing reagents, such as heptafluorobutyric acid, have been used in mobile phases in order to retain these very polar analytes. There is however a reluctance to use these reagents unless absolutely necessary due to the difficulty in clearing them from systems after use. As an example of this problem, it was possible to detect trifluoroacetate adducts in ES negative mode for many months after the removal of trifluoroacetic acid as a component of the reference mass solution. As an alternative to using ion-pairing reagents in the mobile phases, their use in the dissolution solvent only (and therefore at much lower concentrations going into the mass spectrometer source) was investigated. Initially aqueous heptafluorobutyric acid at a concentration of 20 mM was used as a dissolution solvent. A number of compounds, in particular the aminoglycosides and aminocyclitols, showed a significant shift in retention time (for example see figure 5).


Water

1:1 methanol:water

Cyromazine N N

NNH2

NH2

NH

Figure 5. Shift in retention time in presence of ion-pairing reagent - dihydrostreptomycin as example.

However, although retention was improved by inclusion of the ion pairing reagent, in some instances peak shape was poor. For this reason, an alternative column phase, Waters Acquity BEH C18, was investigated. The effect of changing the carbon chain length of the ion-pairing reagent was also investigated. Standards were injected in water containing C4 – C7 perfluoroalkanoic acids and the shift in retention time observed. Figure 6 shows the change in retention time for apramycin. A significant drop in response was observed when perfluoroheptanoic acid was used. The reason for this is unclear but may be a solubility effect. Perfluoropentanoic and perfluorohexanoic acids were selected for further investigation.


20mM HFBA

1:1 methanol:water

DihydrostreptomycinO

OO

O

OH

NHNH2

NHNH NH2

NH

OH

OH

OH

OH

CH2OH

NHOH

OH

None C4 C5 C6 C7

O

OOO

NHOH

O

NH2OH

OH

OH

NH2

OH

OH

NH2

NH2

Apramycin

Figure 6. Shift in retention time with changing chain length of ion pairing reagent.

1.5. Use of alternative ionisation sourcesDuring the course of the project, two alternative ionisation sources (Atmospheric Pressure Chemical Ionisation (APCI) and Atmospheric Pressure PhotoIonisation (APPI)) were made available for the ToF-MS instrumentation in use. Preliminary investigations did not show any significant improvements for the vast majority of the compounds of interest. Further investigatory work was discontinued in the light of these findings.

2. Objective 2. Optimise extraction and clean-up method for compounds of interest.2.1. Optimisation for Protocols 1.1. – 1.3.In order to maintain continuity with the previously developed method, modifications to the extraction and clean-up protocol were restricted to the final aliquoting, drying and redissolution steps. The original protocol was designed to enable additional aliquots to be taken to facilitate this process. In outline this method consists of extraction with oxalic acid in acetonitrile, removal of water with anhydrous sodium sulphate followed by a dispersive solid phase extraction (dSPE) step using a C18 phase. In the standard protocol, an aliquot taken is blown down to dryness and redissolved in 1:1 methanol:water. In this project, further aliquots were taken and redissolved in three different ways:1) water, for analysis by reversed-phase LC-ToF-MS; 2) 9:1 acetonitrile:0.15M ammonium acetate containing 1% formic acid for analysis by HILIC-ToF-MS; 3) 2:1:1 water:phenyl isocyanate in acetonitrile:pyridine (or triethylamine) in acetonitrile, for analysis by reversed phase LC-ToF-MSAs part of the optimisation process, the list of target compounds was rationalised by removal of those compounds either not suited to the proposed procedures (such as closantel, rafoxanide), no longer of interest or for whom a lack of sensitivity in solvent standards indicated that detecting the target concentration was unlikely. This reduced list was then carried forward into the validation exercise.It was noted during the optimisation process that the aminoglycosides and aminocyclitols were not detectable in matrix-matched standards and therefore it was not possible to know whether the extraction and clean-up protocol successfully recovered them or not.

2.2. Optimisation for Protocol 1.4.Aminoglycosides could be detected in matrix-matched standards (bovine kidney) by inclusion of ion-paring reagents (nonafluoropentanoic (NFPA) or undecafluorohexanoic (UDFHA) acid in the final dissolution solvent. However, no aminoglycosides were detected in extracts of spiked samples, indicating that these compounds were not extracted using the generic method.As a precursor to developing a two-stage extraction process, alternative extraction and clean-up protocol were investigated. The results are summarised in Table 3. Inclusion of ion-pairing reagents (Heptafluorobutyric acid, HFBA) in the exaction solvent in place of the oxalic acid did not result in any recovery. Addition of ammonia in the presence of the ion-pairing reagent resulted in low recovery of dihydrostreptomycin and spectinomycin. Changing the acid component to trichloroacetic acid (TCA) resulted in low recovery of two compounds (neomycin B and spectinomycin). Changing the solvent to acetone showed no improvement, but 4 out of 8 compounds were recovered in indicative yields up to 48% using TCA in methanol. This could be further improved to 7 out of 8 with recoveries of up to 87% by changing to ammonia in methanol as the extraction solvent.Alternative phases for the dSPE step were also investigated. Although the effects were small, PSA was found to give slightly better recoveries than NH2 phase.

Table 3. Effect of extraction solvent on aminoglycoside/aminocyclitol recovery from bovine kidneyMeCN – acetonitrile. TCA – trichloroacetic acid. HFBA – heptafluorobutyric acid. MeOH – methanol. NH3 – ammonia solution. NH2 – amino dSPE phase. PSA – primary,secondary amine dSPE phase.


If the extraction solvent for the generic procedure were to be changed, a full re-validation of the procedure for all analytes would have been required. This falls outside the resource allowed for this project. Consequently, a two-stage extraction protocol was proposed, a first extraction using 1% oxalic acid in acetonitrile, identical to the previously developed method, followed by extraction using a solvent more appropriate to the aminoglycosides and aminocyclitols. The first extract would then be cleaned up using the generic protocol whilst the second would be cleaned up by an alternative procedure. The workflow for this process is shown in figure 7.

Figure 7. Workflow for 2-stage extraction and clean-up.

Three alternative procedures were investigated: 1) direct extraction of the residue from the first extraction using 5% ammonia in methanol; 2) dispersal of the residue in water, followed by extraction with 1% ammonia in methanol; 3) dispersal of the residue in water, followed by extraction with 5% ammonia in methanol. As shown in figure 7, best results were achieved using the third extraction protocol. 6 out of the 8 aminoglycosides/aminocyclitols investigated were recovered using this protocol.As a cross check the samples were spiked at 0.1µg kg-1 at the same time with a limited number of analytes that had previously been successfully validated using the generic procedure and the oxalic acid/acetonitrile extracts analysed by LC-ToF-MS. The spiking suite included members of the antifungal, macrolide, quinolones./fluoroquinolones, penicillin, sulphonamide and tetracycline classes. Results matched previous validation experiments.Because of the complexity of the protocols to cover the widest range of analytes (2 extractions, multiple aliquots, up to 6 LC-MS runs using 2 or 3 different columns and conditions), it was decided not to pursue this option any further. However should the need arise to include the aminoglycosides and aminocyclitols in a generic protocol, proof of principle has been demonstrated.

3. Objective 3. Validation of the developed methodSince it was clear from the optimisation experiments that no one protocol was suitable for all analytes, a full validation was carried out in bovine muscle for two protocols: 1) water dissolution; 2) HILIC determination. In addition, single batch validations were carried out for a third protocol, using derivatisation


with phenyl isocyanate/pyridine or phenyl isocyanate/triethylamine. Validation was carried out using a shortened list of 31 compounds as indicated in section 2.1. The full validation consisted of 3 batches of 7 replicate spikes at concentrations of 1, 10 and 100 µg kg-1. Results are summarised in Table 4.

Table 4. Validation data for bovine muscle

To test the ruggedness of the procedures, single validation batches were carried out for bovine kidney and liver (protocols 1 and 2, 10 and 100 µg kg-1 only) and for salmon muscle (protocols 1, 2 and 3-pyridine). The results are summarised in Tables 5 – 7.

Table 5. Validation data for bovine kidney


Table 6. Validation data for bovine liver


Table 7. Validation data for salmon muscle


Dissolution in water is the simplest protocol to implement, requiring no additional reagents and allowing for final analysis using the same conditions as the generic protocol developed in project VM02152. Better limits of detection were achieved for some compounds using HILIC conditions whilst sulfanilamide was only successfully detected using the derivatisation procedure. These three procedures have been validated to provide the opportunity to ‘mix and match’ the protocol used according to the specific needs of the analysis required. Proof-of-principle has been demonstrated for a route by which the aminoglycosides may also be included if required.

4. Evaluate broader applicability of method to remaining analytes in previously developed screening procedure.The design of the extended protocol such that the original protocol is included in its entirety as one aliquot was carried out with the specific intention of maintaining the validity of the data produced in the previous project whilst adding to the range of analytes that can be tested for. Experiments conducted in the course of investigating the use of ion-pairing reagents for dissolution prior to analysis also demonstrated the protocol laws applicable to the previously validated analytes. For this reason, it was not necessary to conduct any further experiments on analytes previously successfully validated, outside of the agreed target analyte list.

5. Transfer methods to surveillance laboratoriesAt the commencement of this project, the main statutory surveillance scheme for the UK (excluding Northern Ireland) was being carried out at another laboratory. Subsequent to the commencement of the project, this work was transferred to Fera. As such, method transfer had to conform to internal transfer procedures. Also it was made clear that there was no requirement in the short and medium term to use this method as a routine procedure for high sample throughput. Consequently, method transfer has been limited to the provision of a draft SOP (see below). This method continues to be used as an investigatory tool for unknown suspect samples and as such remains the purview of the development team. The draft


SOP is available to other laboratories carrying out surveillance for the Veterinary Medicines Directorate.


References to published material9. This section should be used to record links (hypertext links where possible) or references to other

published material generated by, or relating to this project.

[1] Ishii R, Horie M., Chan W., MacNeil J., Multi-residue quantitation of aminoglycoside antibiotics in kidney and meat by liquid chromatography with tandem mass spectrometry. Food Addit. Contam: Part A 2008, 25(12), 1509-1519.[2] Chiaochan C., Koesukwiwat U., Yudthavorasit S., Leepipatpiboon N., Efficient hydrophilic interaction liquid chromatography-tandem mass spectrometry for the multiclass analysis of veterinary drugs in chicken muscle. Anal. CHim. Acta 2010, 682, 117-129.[3] Turnipseed S.B., Clark S. B., Karbiwynk C. M., Andersen W. C., Miller K. E., Madson M. R., Analysis of aminoglycoside residues in bovine milk by liquid chromatography electrospray ion trap mass spectrometry after derivatization with phenyl isocyanate. J. Chromatogr. 2009, 877, 1487-1493.


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