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Available online at www.sciencedirect.com

Journal of Chromatography A, 1175 (2007) 234–241

Determination of the antimicrobial growthpromoter moenomycin-A in chicken litter

Sandra Perez, Brenna E. McJury,Peter Eichhorn, Diana S. Aga ∗

Chemistry Department, The State University of New York at Buffalo, 611 Natural Sciences Complex, Buffalo, NY 14260, USA

Received 28 July 2007; received in revised form 12 October 2007; accepted 19 October 2007Available online 24 October 2007

bstract

This study aimed to develop and optimize a method for the extraction and analysis of moenomycin antibiotics (a.k.a. flavomycin) in corn-basedeed premix and in chicken litter. Moenomycin-A was isolated from chicken litter using pressurized liquid extraction followed by a solid-phasextraction (SPE) clean-up step. The highly lipophilic nature of moenomycin necessitated the use of the less hydrophobic sorbent, C4-based SPEartridge, and a higher temperature elution solvent, methanol at 50 ◦C, in order to obtain satisfactory percent recoveries. After clean-up, the

ample was analyzed by liquid chromatography–electrospray ionization–mass spectrometry (LC–ESI–MS). Various reversed-phase columns werexamined, including C18, CN, perfluorinated C6, and a porous graphitic carbon. The set of conditions that gave the highest separation efficiencyhile still maintaining symmetrical peak shape was the C18 column using the H2O + 0.3% HCOOH and acetonitrile mobile phase. 2007 Elsevier B.V. All rights reserved.

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eywords: Moenomycin; LC–ESI–MS; Chicken litter; Chicken feed

. Introduction

Moenomycin (molecular structure is shown in Fig. 1) is ahosphorus-containing glycolipid compound which is producedy a group of Streptomyces spp. including S. ghanaensis, S.ambergensis, S. geysirensis and S. ederensis. This antibiotichat is also known as bambermycin, flavomycin, and flavophos-holipol, is mainly effective against Gram-positive bacteria,.e., staphylococci and streptococci, because it cannot pene-rate the outer membrane of Gram-negative bacteria. However,ome activity against Pasteurella spp. and Brucella spp. has beeneported [1]. The spectrum of moenomycin is similar to that ofenicillin and to some extent to that of macrolides. It inhibitseptidoglycan synthesis in the cell wall by inhibiting peptido-lycan polymerases through impairment of the transglycolasectivities of penicillin binding proteins leading to lysis and cell

eath [2,3]. There is, however, no cross-resistance between �-actam antibiotics and moenomycin, as they act on differentenicillin binding proteins [4].

∗ Corresponding author. Tel.: +1 716 645 6800x2226; fax: +1 716 645 6963.E-mail address: dianaaga@buffalo.edu (D.S. Aga).

ttratto

021-9673/$ – see front matter © 2007 Elsevier B.V. All rights reserved.oi:10.1016/j.chroma.2007.10.053

Moenomycin is administered to farm animals as a growth pro-oter. The initial use of antibiotics in animal diets during the

ate 1940s arose from the discovery that having the fermentationroducts of Streptomyces aureofaciens (a strain of bacteria) inhe diets of simple-stomached animals, such as pigs and poul-ry, resulted in growth responses. In the next 50 years, the usef antibiotics as feed additives in pig and poultry productionecame virtually universal [5]. Moenomycin is reported to resistegradation when exposed to a variety of enzymes such as phos-hoesterase, glucosidase, and lipase among others [6]. The usef the antibiotics may cause residues to be found in animal prod-cts, and since the antibiotics are not absorbed to a great extent inhe gut of the animal [7], unmetabolized form are excreted intactn the animal wastes. This continuous feeding of antibiotics tonimals may result to the selection of resistant bacteria againstoenomycin and related antibiotics (cross-resistance). Indeed,

he use of antibiotics in livestock production has been linked tohe increased emergence of resistant strains of pathogenic bacte-ia that could potentially impact human health. Multiresistance

gainst other antibiotics may be due to the promotion of resis-ance genes which are located on the same plasmid [8]. Hence,he European Union has proposed phasing out the use of antibi-tics as antimicrobial growth promoter. In December 1998, the

S. Perez et al. / J. Chromatogr. A 1175 (2007) 234–241 235

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uropean Commission banned the use of bacitracin, spiramycin,ylosin and virginiamycin for growth promoters [9]. Since Jan-ary 2006, monensin sodium, salinomycin sodium, avilamycinnd moenomycin have been banned in EU. In the US, the appli-ation of moenomycin is approved for cattle, swine, and poultryt subtherapeutic levels (0.5–20 mg/kg), either alone or in com-ination with ionophore antibiotics such as monensin, lasalocidr salinomycin, to increase the rate of weight gain and improveeed efficiency [10–12].

Therefore, there is a need to carry out studies to understandhe occurrence and the fate of moenomycin residues in the envi-onment. However, this task is not easily achieved because noeference standard was commercially available at the time whenhis work was conducted, and a sensitive analytical method hasot yet been reported in the literature. In the past, analyticalethods for the determination of moenomycin in medicated

eed relied on biological assays [13,14]. Likewise, microbialrowth inhibition assays were also employed for the detection ofntibiotic residues in studies on the dissipation of manure-borneoenomycin in soil environments [15]. Microbiological assays

ack the sensitivity and selectivity necessary for trace analysisn complex matrices. High-performance liquid chromatographyHPLC) combined with ultraviolet (UV) detection has also beensed for the analysis of moenomycin residues in feeds providingetection limits in the low ppm range [16]. However, the use ofV for detection of analytes in complex matrices, such as soilr manure, is problematic because of the high absorbance back-round of co-extracted natural organic matter. To our knowledge,his is the first time that a paper reports the determination of

oenomycin in chicken litter based on LC–ESI–MS.

. Experimental

.1. Chemical standards

The organic solvents acetonitrile, dichloromethane, ethylac-tate, hexane and methanol were A.C.S. grade (Burdick &ackson, Muskegon, MI, USA). Water was prepared with aanopure Diamond water purifier (Barnstead, Dubuque, IA,

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of moenomycin-A.

SA). All chemicals used for preparation of buffers were ofeagent grade.

.2. Isolation and purification of moenomycin from chickeneed premix and estimation of stock solution concentration

A standard solution of moenomycin was prepared by isolat-ng the active ingredient from the medicated chicken feed premixainProTM. To this end, an 11-mL stainless steel extraction cellf the pressurized liquid extraction (PLE) system (ASE 200,ionex, Sunnyvale, CA, USA) was loaded with 2 g of chicken

eed premix. The extraction was performed with methanol at0 ◦C, 500 psi, and two static cycles of 5 min; the flush volumeas 60%, and the purge time was 60 s. The extract of approxi-ately 25 mL was diluted to 250 mL with water and loaded ontoSep-PakTM 500 mg tC18-SPE cartridge (Waters, Milford, MA,SA) preconditioned with 2 × 2 mL of ethyl acetate, 2 × 2 mLf methanol and 2 × 2 mL of water. After drying of the cartridget was washed with 2 × 2 mL hexane/dichloromethane (1:1),× 2 mL ethyl acetate, and 3 × 2 mL ethyl acetate/methanol

3:1). Finally, moenomycin was eluted with 3 × 2 mL methanol.he eluate was evaporated to dryness under a stream of nitro-en and reconstituted with 1 mL methanol. The extract wasurther diluted with 9 mL water and loaded onto a precondi-ioned 6 mL/500 mg strong anion exchange (SAX) cartridgeJ.T. Baker, Phillipsburg, NJ, USA). The dried cartridge wasashed with 2 × 2 mL methanol and 2 × 2 mL 0.6% KCl inethanol. Then, moenomycin was eluted using three 2-mL por-

ions of 1 M sodium acetate. This SPE eluate was freeze-driednd the residue reconstituted with 1 mL methanol. Precipitatedodium acetate was separated from the solution by centrifuga-ion at 13,148 × g for 5 min (Minispin, Eppendorf, Westbury,Y, USA). Purity of the obtained solution was confirmed byC–UV–MS analysis. Aliquots of this methanolic moenomycinolution were diluted (1:10 and 1:100) to determine the concen-

ration using UV absorbance spectroscopy. Absorbances were

easured at a wavelength of 258 nm using an Agilent 8453V-vis spectrophotometer (Palo Alto, CA, USA). A molar

bsorptivity constant of A258nm = 21,357 M−1 cm−1 reported

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36 S. Perez et al. / J. Chrom

reviously [17], was used for calculating the moenomycin con-entration using Lambert–Beer’s Law.

.3. Selection of HPLC column and mobile phaseomposition

Four narrow-bore (100 mm × 2.1 mm I.D.) reversed-phaseiquid chromatography columns were compared in terms of peakymmetry, signal intensity and capacity factor: BetaBasic-C18,etaBasic-CN, Fluophase-RP, and Hypercarb (Thermo Elec-

ron, San Jose, CA, USA). The liquid chromatograph usedas an Agilent Series 1100. Binary solvent systems were used

onsisting of: solvent (A) 0.3% formic acid or 10 mM ammo-ium acetate (pH 7.6), and solvent (B) methanol or acetonitrile,o give four combinations of different mobile phase compo-itions. A generic gradient elution program was used for allolumn/mobile phase compositions: 95% A for 1 min, lin-ar decrease to 5% A within 9 min, hold for 4 min, return tonitial conditions within 0.5 min and equilibrate for 5.5 min.he flow rate was 250 �L/min and the injection volume was0 �L.

.4. Mass spectrometric analysis

The mass spectrometric analysis was performed on an Agilenteries 1100 SL single-quadrupole instrument equipped with anlectrospray ionization (ESI) source. The capillary voltage was4500 V for the positive ion mode and −4000 V for acquisi-

ions in the negative ion mode. Nitrogen was used as nebulizeras (35 psi) as well as drying gas at a temperature of 350 ◦C10 L/min). For quantitative analysis of moenomycin-A, the dataere acquired in selected ion monitoring (SIM) mode detect-

ng the two pseudomolecular ions [M − H]− and [M − 2H]2−t m/z 1580.6 and 789.8, respectively, using fragmentor valuef 140 units. For the acquisition of the mass spectra in full-scanode, fragmentor was set to 120 (low) or 240 units (high). Data

cquisition and processing was done with the software Chem-tation Rev. A.09.03.

.5. Chicken litter analysis

.5.1. Solid-phase extractionExperiments were conducted to compare the extraction

fficiencies of three SPE cartridges, namely, Sep-Pak tC18Waters), Bakerbond C4 and Isolute ENV+ cartridges (both J.T.aker, Phillipsburg, NJ, USA). All cartridges contained 500 mgacking material. Two-hundred fifty milliliter water samplesontaining 10% methanol were spiked with a methanolic moeno-ycin solution at a concentration of 2 �g/L and loaded onto

he cartridges. Elution of the dried cartridges was carried outsing three 2 mL portions of methanol (50 ◦C). The eluates were

vaporated to dryness and reconstituted in 1 mL 25% methanol.alibration curves were constructed in the range from 0.05 tomg/L yielding good linearity (R ≥ 0.998). The limit of quan-

ification for spiked chicken litter was estimated to be 0.02g/kg.

iCaIt

r. A 1175 (2007) 234–241

.5.2. Pressurized solvent extractionExtraction of chicken litter was carried out on the PLE system

sing methanol. An 11-mL stainless steel extraction cell wasoaded with 5 g of chicken litter and filled with diatomaceousarth. The extraction conditions were as follows: 500 psi, twoycles at 4 min each, flush volume 60%, purge time 60 s. Differ-nt extraction temperatures were evaluated. The extract volumef approximately 25 mL was diluted with 225 mL of water andoaded onto the previously selected C4-SPE cartridge. The driedartridge was sequentially washed with 3 × 2 mL of each of (a)ichlorometane/hexane (1:1), (b) ethyl acetate, and (c) ethylcetate:methanol (3:1). Elution of the analyte was completedith 3 × 2 mL of methanol (50 ◦C). The eluate was evaporated

o dryness under a gentle stream of nitrogen and reconstitutedith 1 mL of 25% methanol. The final extract was centrifuged

12,000 × g for 4 min) and the supernatant was injected into theC system.

.5.3. ChromatographySeparations were achieved on a Thermo Hypersil-Keystone

etaBasic-18 100 mm × 2.1 mm (3 �m) column equipped with10 mm × 2.1 mm guard column of the same packing material.he mobile phases were (A) water acidified with 0.3% formiccid and (B) acetonitrile. The gradient program started from 75%/25% B. After 1 min, the portion of A was linearly decreased to% within 10.0 min. These conditions were held for 4.0 min. Thenitial mobile phase composition was restored within 0.1 min and

aintained for column regeneration for another 5.9 min result-ng in a total run time of 20 min. The flow rate was 250 �L/minnd the injection volume was 20 �L.

. Results and discussion

.1. Isolation of moenomycin from chicken feed premix

The chicken feed premix GainProTM is a heterogeneous mix-ure of corn meal containing 2% moenomycin [18] and highmounts of inorganic salts. Due to the heterogeneity of theeed premix a multi-step extraction/clean-up procedure waseeded to isolate moenomycin so as to use it as a referencetandard for further studies. Moenomycin exhibits limited sol-bility in solvents of low to medium polarity; however, higholubilities (g/L range) have been reported for this compound inethanol, and in the aprotic solvents N,N-dimethylformamide

nd dimethylsulfoxide [19]. As methanol has the lowest boil-ng point of these three solvents, it was chosen for the PLE.he raw extract was yellowish-brown indicating co-extractionf other feed premix components. In order to eliminate thesenterferences, a clean-up protocol was established based on awo-step SPE. The diluted raw extract obtained from the PLEystem was therefore loaded on a C18-SPE column. In ordero increase the selectivity of the SPE, a sequential elution withncreasing solvent polarity was performed for the elution of the

18-SPE cartridge using hexane/dichloromethane (1:1), ethylcetate, ethyl acetate/methanol (3:1) and eventually methanol.n a second step, the acid functionalities of moenomycin, i.e.he phosphoric acid diester and the carboxylic acid group, were

S. Perez et al. / J. Chromatogr.

Fig. 2. LC–(−)ESI–MS chromatogram of purified feed premix extract. Scanrange from m/z 100 to 1700. Inset shows the extracted ion chromatograms ofthe [M − H]− (m/z 1581), [M − 2H]2− (m/z 790) and the fragment ion m/z 575.The signal at 11.2 min corresponds to moenomycin-A, while the adjacent peaksaum

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t higher retention times are other members of the moenomycin family. Thenresolved signals eluting between 9.0 and 10.5 min are as yet unidentifiedoenomycins [20].

xploited by means of anion-exchange purification allowingo remove neutral and cationic compounds that had not beenliminated in the previous clean-up step. Again, a sequentiallution of the SAX cartridge was carried out using solventsf increasing ionic strength (methanol, 0.6% KCl in methanol,nd 1 M sodium acetate). Analysis of the final standard solu-ion using UV detection at 246 nm and MS detection in either

he positive or negative ion mode (scan range m/z 100–1700)howed that this solution contained only few interfering com-ounds of co-extracted matter (Fig. 2); the adjacent peaks atigher retention times relative to moenomycin-A (11.2 min)

tMoe

able 1valuation parameters of selected columns for different mobile phases

olumn Retentionfactor

Peak area[M − H]−

Asymmetry factora

[M − H]−Peak[M −

A) H2O + 0.3 % HCOOH (B) acetonitrileC18 9.17 605.3 1.64 1944.Cyano 7.65 513.8 1.25 1442.Fluophase 8.91 397.9 1.11 1129.

A) H2O + 0.3 % HCOOH (B) methanolC18 n.e. – – –Cyano 9.41 247.4 2.94 962.Fluophase 11.98 312.9 1.28 1280.

A) H2O + 10 mM NH4OAc (B) acetonitrileC18 8.07 13.3 1.26 356.Cyano 6.28 18.6 1.00 344.Fluophase 7.55 15.4 1.02 241.

A) H2O + 10 mM NH4OAc (B) methanolC18 n.e. – – –Cyano 7.6 5.92 1.07 71.Fluophase 10.39 10.4 1.09 377.

ignal of moenomycin-A ([M − 2H]2−) used for calculation of plate number. n.e., ana According to IUPAC definition.

A 1175 (2007) 234–241 237

ere other members of the moenomycin family. The unresolvedignals eluting between 9.0 and 10.5 min were as yet unidentifiedoenomycins.

.2. Selection of HPLC column

A liquid chromatographic method was formulated to achievehe highest sensitivity and optimal peak shape for moeno-

ycins. The reversed-phase columns evaluated were identicaln dimension, with packing material of comparable pore sizes,ut differed in the composition of the stationary phase. Twoqueous solvents and two organic solvents were used to opti-ize separation efficiency in each column. Either acetonitrile

r methanol was used as the organic solvent, while either waterith 0.3% formic acid or 10 mM ammonium acetate solutionas used as the second solvent. The purpose of the formic

cid in the aqueous mobile phase was to increase the ioniza-ion efficiency and improve detection by mass spectrometry.s discussed above, only the doubly charged ion was used foruantification. Thus, ammonium acetate was used to increasehe ratio of the doubly charged analyte to the singly chargednalyte.

The selection of the column and mobile phase was basedn the following parameters: peak area, peak shape/symmetry,atio of the doubly charged to the singly charged species,nd the number of theoretical plates (N) (Table 1). Only oneon, the doubly charged species of moenomycin-A, was usedor the calculations compiled in Table 1. The Hypercarb col-mn results were omitted from the table due to the fact thatoenomycin did not elute from the column at a reasonable

ime (no elution up to 60 min of chromatographic run time).oenomycin-A proved to be the best ion (among all the

ther moenomycins) to monitor in order to compare the differ-nt chromatographic conditions, because moenomycin-A has

area2H]2−

Asymmetry factor[M − 2H]2−

Area ratio[M − 2H]2−/[M − H]−

Plate number(/1000)

2 1.61 3.21 21.08 1.30 2.81 28.54 1.14 2.84 32.9

– – –1 3.12 3.89 2.331 1.33 4.09 40.4

7 1.25 26.8 25.88 1.05 18.6 28.49 1.04 15.7 29.8

– – –8 3.03 71.8 6.455 1.07 36.3 37.3

alyte not eluting from column.

238 S. Perez et al. / J. Chromatogr. A 1175 (2007) 234–241

F [M −e moni

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ig. 3. (A) LC–(−)ESI–MS extracted ion chromatograms of moenomycin-A (xtracted ion chromatograms of moenomycin-A ([M − 2H]2−) using 10 mM am

he most abundant molecular ion and was easily detected atll the conditions tested. The set of conditions with the besteak area was obtained using the BetaBasic-C18 column and aobile phase consisting of H2O + 0.3% HCOOH and acetoni-

rile. The best peak shape was obtained using the BetaBasic-CNolumn and an ammonium acetate and acetonitrile mobilehase (Fig. 3). The highest ratio of doubly charged to singlyharged was found using a BetaBasic-CN column and an ammo-ium acetate and methanol mobile phase. A mobile phaseonsisting of ammonium acetate and methanol coupled with aluophase-RP column produced the greatest number of theoret-

cal plates (Table 1). Although most conditions produced highlyymmetrical peak shape, a higher priority was placed on peakrea. By giving the highest area and intensity, the detection limitould be lowest, and therefore most applicable to trace analysisf moenomycin residues. Fig. 3 demonstrates the considerableifferences in the areas and peak heights that were obtainedsing the different columns and mobile phases. The slight differ-nces in peak shape are much less distinct. The retention times ofoenomycin-A varied between 8.3 and 12.7 min. Methanol was

nable to elute moenomycin from the BetaBasic-C18 column.ue to the fact that the retention time did not vary consider-

bly, the time of analysis did not affect the selection of the bestonditions. Thus, the set of conditions that were determined to

ive the highest overall separation efficiency while still main-aining symmetrical peak shape and area was the BetaBasic-C18olumn using the H2O + 0.3% HCOOH and acetonitrile mobilehase.

nafb

2H]2−) using H2O + 0.3% formic acid as aqueous phase. (B) LC–(−)ESI–MSum acetate as aqueous phase.

.3. Mass spectrometric characterization

The mass spectrometric detection of moenomycin-A was fea-ible in positive and negative ion mode using an ESI source. Theass spectra acquired over a scan range from m/z 300 to 1700

re displayed in Fig. 4 for a low fragmentor voltage (no in-ource CID induced) and high fragmentor voltage (leading ton-source CID). In the (+)ESI mass spectrum corresponding tohe low fragmentor voltage, a protonated molecule, [M + H]+, at/z 1582.7 as well as a mono-sodium adduct, [M + Na]+, at m/z604.7 were observed (Fig. 4A). The detection of a large num-er of fragment ions indicated the instability of the molecularon(s). The mass spectral assignments of all major fragment ionseriving from the cleavage of the protonated molecule have beenescribed in an ion trap mass spectrometer with an ESI sourceESI–IT–MS) in a previous work [20]. The MS2 experimentn [M + H]+ had generated most of the fragment ions detectedn the present work using a single-quadrupole instrument. Thessignment of the fragment ions is summarized in Table 2 reveal-ng that aside from the loss of the centrally attached D-ring, atepwise cleavage of the molecule occurred starting from theerminal hydrocarbon chain. All of the fragment ions containedhe three moieties A, B and C suggesting that the positive chargeas preferably located within these building blocks. It is worth

oting that under the applied conditions moenomycin-A formeddoubly charged molecular ion of the type [M + 2H]2+, of which

ragmentation at low fragmentor voltage gave rise to two dou-ly charged product ions at m/z 621.8 and 528.8 (Fig. 4B). The

S. Perez et al. / J. Chromatogr. A 1175 (2007) 234–241 239

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ig. 4. Mass spectra of moenomycin-A under positive (left side) and negativelower panels).

ass spectrum acquired at a high fragmentor voltage differedrom the one recorded at the low fragmentor voltage by the sup-ression of the three doubly charged species. The sodium adductf moenomycin-A, in turn, did not undergo fragmentation to anyubstantial extent as reflected by the absence of fragment ionsxpected at m/z values 22 units higher than those observed forhe protonated molecule. With respect to employing the posi-ive ion mode for quantitative trace analysis of moenomycin-An complex samples, the extensive fragmentation of the pro-

onated molecule and the concurrent formation of the sodiumdduct rendered this mode unsuitable. Attempts made to pro-ote the sodium cationization by replacing formic acid in theC mobile phase with micromolar concentrations of sodium

wvw

able 2ssignment of fragment ions of moenomycin-A

ositive ion mode

/z Ion

604.7 [M + Na]+

582.7 (792.3) [M + H](2)+

420.7 [M + H − D]+

242.5 (621.8) [M + H − I](2)+

080.5 [M + H − (D,I)]+

056.5 (528.8) [M + H − (G–H–I)](2)+

824.3 [M + H − (F–G–H–I)]+

662.3 [M + H − (D,F–G–H–I)]+

459.2 [M + H − (D–E–F–G–H–I)]+

he values in parentheses correspond to the doubly charged species.

side) ionization mode using high (upper panels) and low fragmentor voltages

cetate were successful in terms of suppressing the protonationf the molecule. Yet the high affinity of the carbohydrate residueso the alkali ion in conjunction with the presence of two acidicrotons (phosphoric acid diester in portion G and carboxyliccid in portion H) led to the production of not only the mono-odiated species [M + Na]+ but also to higher adducts of the typeM + 2Na − H]+ and [M + 3Na − 2H]+, thereby strongly limit-ng the applicability of this approach for quantitative purposesspectrum not shown).

As for the application of the (−)ESI mode, the mass spectraere comparatively simple (Fig. 4C and D). At a low fragmentoroltage the base peak corresponded to [M − 2H]2− (m/z 789.8),hereas the ion of the deprotonated molecule was observed at

Negative ion mode

m/z Ion

1580.6 (789.8) [M − H](2)−1537.7 (768.3) [M − H − OCNH](2)−

575.6 [M − H − (H–I)]2−554.3 [M − H − (OCNH, H–I)]2−

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nafafpsfnin line with increasing amounts of UV-absorbing substancesextracted at higher temperatures (Fig. 5). The chromatogramsof the UV traces at 254 nm corresponding to the four testedextraction temperatures reflect the enhanced solubilization of

40 S. Perez et al. / J. Chrom

/z 1580.6 (Table 2). The latter species produced a fragment ionf low signal intensity at m/z 1537.7. This elimination of a neutralragment with 43 Da had also been observed in (−)ESI–IT–MSnd had been assigned as the loss of OCNH out of the car-amate substituent on the F-ring [20]. For the doubly chargedolecular ion (m/z 789.8) that underwent the same fragmenta-

ion (Fig. 4C and D), this expulsion translated into an apparentoss of 21.5 Da giving rise to the ion m/z 768.3. The increasef the fragmentor voltage led to the generation of two furtherragment ions at m/z 575.6 and 554.3 (Fig. 4C). The mass differ-nce of 21.5 Da between these two ions indicated that they botharry two negative charges. The ion m/z 575.6 corresponded tohypothetical singly charged fragment ion with a m/z 1152.2.his very fragment ion represented the base peak ion in the MS2

pectrum of [M − H]− recorded on the IT–MS [20]. It is derivedrom the cleavage of the ester bond between the phosphoric acidiester (G) and the carboxy-glycol (H) (see Fig. 1). In view ofhe outcomes of the comparisons between positive and negativeon mode, the latter was selected for the quantitative analysis of

oenomycin-A in chicken litter extracts.

.4. Optimization of solid-phase extraction and pressurizediquid extraction for the analysis of moenomycin in chickenitter

For the determination of moenomycin-A from chicken litter,LE with methanol and subsequent clean-up on SPE cartridgesas used as this combination proved successful in isolatingoenomycin from medicated chicken feed premix (see above)

20]. Here, the first step in proposing a quantitative approachor the analysis of moenomycin-A in chicken litter consisted ofvaluating the suitability of different SPE sorbents in the clean-p procedure. For this purpose, moenomycin-A was spiked inriplicate samples at a concentration of 2 �g/L into 250 mL ofater/methanol (90:10). This solvent mixture composition wassed because it corresponds to a solution obtained when dilut-ng a methanolic chicken litter extract (after PLE) of about5 mL with water to a final volume of 250 mL. After loadinghe spiked sample onto a SepPak tC18 cartridge, the analytesere eluted with methanol. Analysis of the extract after solvent

xchange to water/methanol (75:25) yielded recoveries of 2%.irect analysis of the SPE eluate showed no traces of the ana-

yte indicating that moenomycins were not desorbed from thePE cartridge. Desorption of moenomycin-A, which was appar-ntly very tightly bound through hydrophobic interactions to the18-modified silica, was facilitated by using methanol preheated

o 50 ◦C for elution. This procedure increased the recovery to1 ± 2.3%). In order to weaken the strength of the hydrophobicetention and thus to enable the release of the target compound, aorbent based on C4-modified silica was tested affording recov-ries of as high as 88 ± 2.2%. In an additional experiment, theuitability of the polymeric sorbent Isolute ENV+, a cross-linkedydroxylated polystyrene–divinylbenzene copolymer, was eval-

ated. Recoveries for spiked 250-mL water samples with 10%ethanol amounted to 62 ± 0.5%. Based on these findings,

he C4-modified silica sorbent was considered the best choicemong the tested SPE materials.

Fo

r. A 1175 (2007) 234–241

In the next stage, recovery studies with real chicken litteramples were carried out including the PLE extraction and sub-equent clean-up steps. Therefore, litter samples from untreatedhickens were spiked with moenomycin-A at a concentrationf 0.1 mg/kg. The extractions were performed with PLE attemperature of 50 ◦C followed by sample clean-up on C4-

PE cartridges. The final sample extracts were injected intohe LC–ESI–MS recording both the singly and doubly chargedons of moenomycin in SIM mode. For external quantifica-ion the areas of both species were summed up and plottedgainst the concentrations running from 0.05 to 5 mg/L. Com-arison of the relative ion intensities revealed that the ratioM − H]−/[M − 2H]2− in the aqueous standard solutions wasbout three times lower than the one observed in the chicken litterxtract, representing a possible source for inaccurate quantifi-ation. To address this issue, matrix-matched standard solutionsere prepared in chicken litter extracts, which had undergone

he same analytical procedure as the fortified samples, and com-ared with the matrix-free calibration standards. It turned outhat the slope of the matrix-free calibration curve was aboutour times higher than the one created with matrix-matched stan-ards. Thus, using external calibration without matrix-matchedtandards would result in substantial underestimation of theecoveries.

Overall, the chemical composition of the matrix had a sig-ificant impact on the response factors, hence requiring criticalssessment when it comes to quantitative measurements. Theact that moenomycin-A occurred in a singly and doubly chargednionic form is likely to be not only susceptible to interferencesrom co-extracted matrix components during the ionizationrocess in the ESI interface, but also to differences in ionampling and transmission of the two species differing by aactor of two in the m/z values. That co-extracted matrix compo-ents had a marked impact on the relative ion abundances was

ig. 5. HPLC–UV chromatograms (254 nm) corresponding to purified extractsbtained at different extraction temperatures.

S. Perez et al. / J. Chromatogr.

F(C

mefmm(nubt

4

pfitrtsocFim[t

fcvpmf

A

SfimteE

R

[[

[[[[[

ig. 6. Recoveries of moenomycin-A from spiked chicken litter at 100 �g kg−1

N = 3). Extracts obtained after pressurized liquid extraction and clean-up on

4-SPE cartridge.

atrix interferences with increasing solvent temperature in thextraction cell. Comparison of the extraction efficiencies at theour different temperatures (each quantification with its set ofatrix-matched standards) showed the highest recoveries ofoenomycin at a temperature of 50 ◦C amounting to 55 ± 0.3%

N = 3). Increasing the PLE temperature to 75 or 100 ◦C didot improve extraction efficiency, but led to lower recoveriesnder both conditions (Fig. 6), possibly owing to thermal insta-ility of the analyte. Carrying out the extraction in turn at roomemperature likewise yielded lower recoveries.

. Conclusions

In this paper, we described a method for the isolation andurification of moenomycin from chicken feed premix. The puri-ed moenomycin was used in the development of a protocol

o determine moenomycin antibiotic residues in chicken litter,elying on PLE, clean-up by solid-phase extraction, and quanti-ation by LC–(−)ESI–MS. This research demonstrated that thetrong hydrophobic nature of moenomycins necessitates the usef an SPE sorbent with lower hydrophobicity (C4-SPE cartridge)ombined with hot methanol elution for increased recoveries.or the trace analysis of moenomycins by LC–(−)ESI–MS it

s important to use the negative ionization mode at low frag-entor voltage in order to obtain higher signal intensity for

M − H]− and [M − 2H]2− by minimizing the fragmentation ofhese molecular ions. Finally, results show that detection limits

[[

[[

A 1175 (2007) 234–241 241

or moenomycin are influenced significantly by the mobile phaseomposition (with ACN being found to be the best organic sol-ent) and the stationary phase of the HPLC column. The methodresented in this paper can be applied to the determination ofoenomycins in environmental samples (manure or soil) and in

eed samples (corn).

cknowledgements

This material is based upon work supported by the Nationalcience Foundation under grant no. 0233700. Any opinions,ndings, and conclusions or recommendations expressed in thisaterial are those of the authors and do not necessarily reflect

he views of the National Science Foundation. S.P. acknowl-dges a post-doctoral fellowship from the Spanish Ministry ofducation, Culture and Science (EX2003-0687).

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