BIOLOGICAL MASS SPECTROMETRY, VOL. 21, 675-687 (1992)

Determination of Erythromycin A in Salmon Tissue by Liquid Chromatography with Ionspray Mass Spectrometry? Stephen Pleasance,$§(IJohn Kelly,§ M. Denise LeBlanc, Michael A. Quilliam and Robert K. Boyd$ Institute for Marine Biosciences, National Research Council, 1411 Oxford Street, Halifax, Nova Scotia B3H 321, Canada

David D. Kitts Department of Food Science, Faculty of Agricultural Sciences, University of British Columbia, 6650 NW Marine Drive, Vancouver, British Columbia V6T 124, Canada

Keith McErlane Faculty of Pharmaceutical Sciences, University of British Columbia, 2146 East Mall, Vancouver, British Columbia V6T 123, Canada

M. Ruth Bailey and David H. North Health Protection Branch, Health and Welfare Canada, Dartmouth, Nova Scotia B2Y 327, Canada

A reverse-phase liquid chromatography/mass spectrometry (LC/MS) method, incorporating gradient elution, is described for the characterization of residual erythromycin A and its metabolites in salmon tissue. The method uses ionspray, a mild atmospheric pressure ionization technique which provides an abundant protonated molecule well suited for selected ion monitoring experiments. Tandem mass spectrometry (MSWS) using collision-induced dissociation was used to provide structural information. The LC/MS method was tested for the analysis of salmon tissue spiked with erythromycin A at levels between 0.01 and 1 p.p.m. A simple extraction and clean-up procedure, slightly modified from that described by Takatsuki et ul. (J. Assoc. Off. A n d . Chem. 70, 708 (1987)), was used in this work, Using selected ion and selected reaction monitoring techniques, the LC/MS and LC/MS/MS methods provided detection limits of < 10 and 50 ng g- respectively. Confirmatory full-scan LC/MS and LC/MS/MS spectra were obtained at the 0.5 and 1 pg g-' levels, respectively. Using a combination of these techniques, the presence of residual erythromycin A was confirmed in the tissue of fish administered medicated feed containing the antibiotic. In addition, several metabolites and degradation products of erythromycin A, including anhydroerythromycin and Ndemethylerythromycin, were detected and where possible confirmed by comparison with authentic compounds. Although this analytical method has been shown to afford the necessary sensitivity and precision, application of these techniques to high-throughput quantitative analyses will require development of an improved clean-up procedure and preferably also of a suitable surrogate internal standard.

',

INTRODUCTION Antibiotics are used extensively in the intensive farming of terrestrial livestock to maintain optimal health and promote growth. More recently, the worldwide increase in aquaculture has also seen an increasing use of antibiotics in fish such as salmon. Unfortunately, the use of these drugs can leave residues in edible tissue. These drug residues may have direct toxic effects on consumers, e.g. allergic reactions, or indirectly cause problems through the induction of resistant bacteria. Therefore, in addition to establishing conditions of use with regard to efficacy and safety of the animal species, tolerance levels must also be established and regulated to assure human food safety.

t NRCC no. 34825.

5 Authors to whom correspondence should be addressed.

Yj Under contract from SCIEX, Thornhill, Ontario, Canada.

11 Present address: The Wellcome Foundation, Department of Bioanalytical Sciences, South Eden Park Road, Beckenham BR3 3BS, UK. 1052-9306/92/120675-13 $1 1S O

0 1992 Crown copyright Canada

Erythromycin A (EA, Fig. 1 and Table 1) is a member of the macrolide group of antibiotics, which consist of a 12-, 14- or 16-membered macrocyclic lactone to which sugar moieties, including amino and deoxy sugars, are attached.' All are produced by various Streptomyces strains, and are used in veterinary practice to treat not only infections by Gram-positive bacteria, but also to improve growth or feed efficiency. The structures, names and convenient acronyms, for erythromycin A and related compounds,',' are shown in Table 1 and Fig. 1. Most antibiotics, including the macrolides, are now assayed by microbiological methods. While these methods are suitable for the routine screening of drug residues in food products, they are often lengthy and lack the specificity and precision for regulatory purposes. The rnacrolides themselves present a significant challenge for instrumental analysis due to their polarity, high molecular weight, acid lability and lack of a significant chromophore. Chromatographic methods, including thin-layer chr~matography,'-~ and liquid chromatography with ~ l t r a v i o l e t ~ fluorescence6 *~ and Received 30 June I992 Accepted 20 August 1992

S. PLEASANCE ET A L

616

R1,

N I

P

3

CH3 CH3

1

0

a

C

b

d

e

Figure 1. Structures of aglycone moieties a-e and sugar residues in EA and related compounds; see Table 1 for compound names and abbreviations, for specific combinations of a+ and substituents R,-R, .

Table 1. Erythromycin A and related compounds Name

AQlycone

Code

Erythromycin A Erythromycin B Erythromycin C Erythromycin D Erythromycin F N- Demethylerythromycin A Erythromycin A-N-oxide Erythromycin A enol ether (8.9-anhydroerythromycin A-6.9-hemiketal) N-Demethylerythromycin A enol ether Anhydroerythromycin A (erythromycin A-6,9;9,12-spiroketal) Anhydro-N-demethylerythromycin A (N-demethylerythromycin A-6,9;9,12-spiroketal) Anhydroerythromycin C (erythromycin C-6,9;9,12-spiroketal) Pseudoerythromycin A 6,9- hemiketal N - Demethylpseudoerythromycin A 6,9-hemiketal Pseudoerythromycin A enol ether (8.9-anhydropseudoerythromycin A 6,g-hemiketal) N - Demethlpseudoerythromycin A enol ether

a a a a a a a b

€A EB EC ED EF dMeEA EANO EAEN

OCH, OCH, OH OH CH3 OCH, OCH, OCH,

b C

dMeEAEN AEA

C

AdMeEA

C

d d

d M epsEA HK

e

DsEAEN

e

dMeps EAEN

n.a., not aDDliCable

R,

Mol wt

OH H OH H OCH, OH OH OH

H H H H OH H H H

733 717 719 703 749 719 749 715

OCH, OCH,

OH n.a.

H H

701 715

OCH,

n.a.

H

701

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OH

ma.

H

701

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OCH, OCH, OCH,

n.a. n.a. n.a.

H H H

733 719 715

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H

701

82

H

H

R3

ERYTHROMYCIN BY LC/MS

electrochemical7 detection, have been described for the determination of erythromycins and other macrolide antibiotics. However, applications of these methods to residues in food products have been limited. For confirmatory analyses, mass spectrometry would be the preferred method of detection. The conventional electron impact (EI)' and chemical ionization (CI)9 mass spectra of several macrolide antibiotics, including erythromycin A, have been described. More recently a method for the determination of residual erythromycin A in pork and beef, using combined gas chromatography and mass spectrometry (GC/MS), has been reported."." While demonstrating adequate sensitivity the method required conversion of erythromycin A, via acid hydrolysis and acetylation, to erythralosamine acetate prior to analysis by GC/MS. Extensive clean-up of the extract was also required, to remove interferences for detection and quantitation by selected ion monitoring of a relatively low mass ( m / z 200), and thus non-specific, fragment ion. While liquid chromatography combined with mass spectrometry (LC/MS) would be the ideal analytical tool for the analysis of antibiotics in biological matrices, it is only in recent years that LC/MS has become more widely used and gained greater acceptance as a viable routine analytical technique. Thermospray (TSP),' one of the first commercial LC/MS interfaces, has been used with limited success for the analysis of antibiotic residues.13 Some of the general criticisms made of LC/TSP/ MS include its wide variation in response with compound class, insufficient signal stability for quantitative analyses, and the frequent lack of structurally useful fragment ions, though special ancillary techniques can sometimes overcome this problem to some extent. This is particularly important in residue analysis where more than one ion is generally required to validate analyte identity. The capability of the particle-beam (PB)I4 interface to generate conventional, library-searchable EI mass spectra has generated much recent interest. This interface was recently evaluated for the LC/MS analysis of several pharmaceutical agents, including some representative antibiotics used in agriculture.' While the authors confirmed that informative EI spectra could be obtained for the more stable and volatile compounds examined, they i n d i ~ a t e d ' that ~ compromises in LC conditions may be required for optimal performance of the interface. Full-scan EI-PB detection limits of the order of 100 ng were reported15 for most of the drugs examined (which unfortunately did not include any macrolides). In general, problems with analyte volatility and thermal stability encountered in GC analysis are also encountered in the generation of EI, and to a lesser extent CI, mass spectra. These problems are not applicable to LC, and this advantage justifies the continuing search for an effective LC/MS interface which does not involve EI or CI mass spectrometry. Thus, in a recent comparison of LC/MS interfaces for the analysis of carbamate pesticides in vegetables," LC/PB/MS was found to be incapable of providing sufficient sensitivity or reproducibility for the reliable detection or quantitation of the target compounds at the legal tolerance levels. One of the potential advantages of the PB interface,

671

however, is that it may be used in conjunction with softer ionization techniques such as fast atom bombardment (FAB). While a FAB mass spectrum of EA obtained via a PB interface was described recently,' only flow injection analysis was attempted and 5 pg of the antibiotic was required. The application of LC/ FAB/MS using a continuous-flow (CF) interface, to the determination of erythromycin 2'-ethylsuccinate in plasma, has recently been rep~rted.''.'~These authors compared two methods of reduction of flow rate from that used with conventional columns (1 ml min-') to that compatible with CF-FAB (3-5 pl min-'). The techniques used were post-column splitting and a complex phase-system switching technique in which the analyte was enriched on a trapping column after separation. While t h e ~ e ' * ~and ' ~ other workers have shown that the technique is an effective LC/MS interface for selected applications, LC/CF-FAB/MS does not appear to have the inherent sensitivity, the analytical ruggedness, nor the ease of use necessary for high-throughput regulatory residue analysis. Due largely to the efforts of Henion and his collaborators,20-22 atmospheric pressure ionization (API) is currently showing great promise as the key to a universal mass spectrometry interface for LC and other separation techniques operated at or near room temperature. Atmospheric pressure chemical ionization using a corona discharge has been used to generate mass spectra of several macrolides including EA.23 In a more recent several commercial LC/MS interfaces were compared for the analysis of a variety of antibiotics used in the aquaculture industry. The particular API technique of ionspray2' (ISP, pneumatically assisted electrospray) was found to provide excellent sensitivity for these compounds. Stemming from this comparative the use of LC/ISP/MS for the determination of sulfonamides in fin-fish tissue was reported? Highly promising preliminary results have also been reportedz6 on the application of capillary electrophoresis, coupled on-line to ionspray mass spectrometry, for analysis of a variety of antibiotics including erythromycin. In the present work LC/ISP/MS methodologies are applied to the determination of residual EA in extracts of salmon flesh, and to characterization of its metabolites and breakdown products.

EXPERIMENTAL Chemicals EA was obtained from the Health Protection Branch drug repository, Health and Welfare Canada (HWC), Toronto, Ontario. Anhydroerythromycin A (AEA), erythromycin C (EC) and anhydroerythromycin C (AEC) were obtained from Dr E. G. Lovering, Bureau of Drug Research, HWC, Ottawa, Ontario. Stock solutions of all compounds were prepared by dissolving accurately weighed amounts in methanol. The N-oxide of erythromycin A (EANO), and erythromycin A enol ether (EAEN), were prepared from EA by a slightly modified version of the procedure described by Flynn et al.;" the structure of the purified product was con-

678

S. PLEASANCE ET AL.

firmed by 500 MHz 'H nuclear magnetic resonance (NMR) spectroscopy (Bruker AMX 500). Formic acid was obtained from BDH Chemicals (Poole, UK). OmnisolvTM,acetonitrile, methanol and hexane were purchased from BDH Inc. (Toronto, Ontario). Glassdistilled grade methylene chloride was obtained from Burdick and Jackson (Baxter Corp., Muskegon, Michigan). A Milli-Q water purification system (Millipore Corp., Bedford, Massachusetts), equipped with ion-exchange and carbon filters, was used to further purify glass-distilled water.

organic layer was drained into another separatory funnel and the aqueous layer re-extracted with a further 100 ml of methylene chloride. The combined organic layers were washed with 100 ml of a 10% sodium chloride solution and then dried by filtering through Na,SO,. The solvent was removed by rotary evaporation and the residue redissolved in 5 ml methanol to give a final extract concentration of 5 g tissue equivalent per milliliter. Tissue samples from fish used in the feeding study, and samples of the medicated feed, were also extracted in the same way.

Biological samples

LCMS

Control salmon were purchased from local markets by HWC Health Protection Branch inspectors. Chinook salmon (Oncorhynchus tshuwytschu) used for the feeding study were maintained in a circular flowing seawater tank as described previously.28 Medicated feed (Extruded New Age Salmon Feed, Moore-Clarke, Vancouver, British Columbia) was administered twice daily such that EA was ingested at a rate of 80 mg kg-' of fish per day. Fish used in the present work were removed and killed on day 9 of medication. Twentyfive-gram samples of salmon tissue, assembled from several portions of the carcass, were weighed and stored at - 11 " C ; although this large sample size inevitably resulted in a correspondingly large final volume of extract solution (5 ml, see below), this sampling protocol is essential if the analyzed flesh is to be truly representative of the entire fish. Indeed, a regulatory protocol would demand multiple grindings and mixings of the entire edible flesh followed by a 25 g subsampling of the ground material, but this rigorous procedure was not possible in the present work since only 25 g total were available to us.

All LC/MS analyses were performed using a HewlettPackard 1090 series I1 liquid chromatograph (HewlettPackard, Palo Alto, California), equipped with a ternary DR5 solvent delivery system and an autosampler. This liquid chromatograph was coupled to an API I11 triple-quadrupole mass spectrometer (SCIEX, Thornhill, Ontario), equipped with an atmospheric pressure ionization (API) source and an IonSprayTM interface. A 250 x 4.6 mm i.d. base-deactivated, reverse-phase ZorbaxO Rx-C8 column, protected by a guard column of the same phase, was used throughout this investigation. An injection volume of 25 pl was used. Aqueous acetonitrile containing 0.2% formic acid was used as the mobile phase, at a flow rate of 1 ml min- '. Separations were achieved with a linear gradient of 5% to 65% acetonitrile in 15 min, followed by a hold of 5 min and a post-time of 10 min. A post-column split of 1 : 100 was used, such that lop1 min-' were directed to the API source. A Macintosh IIx computer was used for mass spectrometer control, data acquisition and data processing. The ISP needle voltage was maintained at approximately 5.4 kV, and high-purity air was used as the nebulizing gas at an operating pressure of 80 p.s.i. (approximately 0.6 1 min- '). Tandem mass spectrometric (MS/MS) measurements were based on collisioninduced dissociation (CID) within the r.f. only quadrupole at a collision energy of 40 eV (laboratory frame). Argon was used as the target gas at an indicated thickness of 3.8 x lo', atoms ern-,. For full-scan analyses, including MS/MS .product ion scans, the instrument was scanned from m/z 100 to 800 with a dwell time of 8 ms Da-'. For selected ion and selected reaction monitoring experiments dwell times of either 400,200 or 100 ms per ion/reaction were used.

Extraction of salmon tissue

The extraction procedure used in this investigation was modified from that described by Takatsuki and coworkers."." Briefly, 25 g of chopped salmon tissue were placed in a 250 ml centrifuge bottle with 70 ml of acetonitrile and homogenized for 1 min (PolytronTM, Brinkman Instruments, Rexdale, Ontario). Tissue samples were spiked, prior to homogenization, with 500 pl of EA in solution at various concentrations, to produce the desired levels of fortification. Individual homogeneates were centrifuged at 2000 r.p.m. for 10 min, and the supernatant decanted through fluted filter paper into a 250 ml separatory funnel. The residual pellet was re-extracted with a further 70 ml of acetonitrile, and the combined filtered supernatants extracted with 60 ml of hexane. The acetonitrile layer was drained into a 500 ml separatory funnel to which was added 6.6 ml of a 1 N sodium hydroxide solution followed by 100 ml of methylene chloride with gentle swirling. After the addition of 1 0 0 ml of 1% disodium hydrogen phosphate (Na,HPO,. 12H,O; 0.4% as Na,HPO,), the mixture was shaken vigorously. Sodium chloride (5-10 g) was then added, and after rocking the mixture was set aside for the layers to separate. The

RESULTS AND DISCUSSION Mass spectrometry and MS/MS using flow injection

The structure of EA (Fig. 1 and Table 1) consists of a 14-membered macrocyclic lactone to which desosamine (an amino sugar, 159 Da) and cladinose (a neutral sugar, 160 Da) are attached via glycosidic linkages. Several related erythromycins have been reported in fermentation residues29and in commercial preparations of the antibiotic,' and biologically inactive degradation

ERYTHROMYCIN BY LC/MS

679

content of the mobile phase was found to have only a minor effect on the ISP response of EA, or indeed on that of any of the antibiotics examined in this work and previ~usly.'~ This independence of mobile phase composition is particularly important for gradient elution operation in LC/MS (see below). The fact that the majority of the ion current associated with the analyte is carried by one ion ( M H + ) makes the technique particularly well suited for use with selected ion monitoring techniques. As discussed above with reference to TSP, the lack of abundant characteristic fragment ions can be a disadvantage in residue analysis in the context of validation of analyte identity. The lack of structural information can be remedied by using CID of the MH' ion. This can be accomplished in two ways using the ionspray interface. The first approach makes use of a characteristic of the particular API source used in this investigation, in which controlled CID in the highpressure expansion region after the sampling orifice is

products, such as various anhydroerythromycins (Fig. 1 and Table l), are formed under slightly acidic or neutral aqueous condition^.'^'^^.^^ The labile character of EA under acidic conditions was a concern throughout the present work. The ionspray mass spectrum of EA shown in Fig. 2(a) is not background subtracted, and was obtained by flow injection analysis at 50 p1 min-l using aqueous acetonitrile (50: 50) containing 0.2% formic acid; this spectrum represents consumption of 50 ng of material. Despite the fragility of the molecule under the acidic conditions required for protonation in solution, as a necessary prerequisite for the mild ion evaporation ionization process believed3' to operate in ISP, EA yields a very simple mass spectrum (Fig. 2(a)) containing only the protonated molecule (MH') at m/z 734.4. In particular, no evidence for H,O loss from EA nor for other degradation reactions was apparent, so the time-scale for such reactions must have been appreciably longer than that characterizing the ffow injection analysis. The organic

734

734

576

-716

158 I00

7-00

300

400

500

600

700

mh

Figure 2. Positive ion ionspray mass spectra of EA obtained with an orifice voltage of (a) 50 V and (b) 80 V, with all other interface potentials held constant; annotated m/z values are truncated, not rounded off. (c) Product ion (MS/MS) spectrum of the MH' ion of EA (m/z 734.4); collision energy 40 eV (laboratory frame) using argon as target gas at a thickness of 3.5 x I O l 4 molecules c w 2 . Conditions: 0.5 PI injections of EA (0.1 mg ml-') into a mobile phase of aqueous acetonitrile (50: 50) containing 0.1 'YO formic acid, at a flow rate of 50 pI min-'.

680

S. PLEASANCE ET AL.

exploited, in normal operation, to remove solvent molecules clustered to analyte ions. By varying the potential difference between the sampling orifice and the r.f. only quadrupole which funnels the ions from the interface region to the mass filter, the extent of this CID process can be controlled. The ISP mass spectrum of EA shown in Fig. 2(b) was acquired under conditions identical to those used to obtain Fig. 2(a), except that the orifice potential was changed from 50 V and 80 V, thus increasing the collision energy. The resulting change in the spectrum is dramatic; the MH' ion, while still the base peak, is accompanied (Fig. 2(b)) by an intense fragment ion at m/z 576 (loss of cladinose with simultaneous hydrogen transfer), and both of these ions exhibit several less intense satellite ions due to successive losses of water molecules (18 Da). Another prominent ion appears (Fig. 2(b)) at m/z 158; this is most reasonably assigned to an even-electron ion corresponding to the dehydro-form of the protonated amino sugar (desosamine), formed by cleavage of the glycosidic linkage with simultaneous hydrogen transfer to the macrolide ring.31 It is much less likely that this fragment ion at m/z 158, formed by low-energy CID, could be attributed to an odd-electron ion derived from the cladinose residue. This means of effecting CID is extremely eft% cient, although at yet higher orifice potentials the total ion current associated with the analyte drops off dramatically. The second, more conventional, means of producing CID is via MS/MS, in which collisions of a massselected precursor ion (MH' in the present work) with a target gas occur in a collision cell (in this case an r.f. only quadrupole) between two mass analyzers. The MS/MS product ion spectrum of m/z 734.4, thus obtained using argon as the target gas, is presented in Fig. 2(c). The fragment ions observed are almost identical to those seen in Fig. 2(b). Both spectra are remarkably similar to a recently reported high-energy CID spectrum of protonated EA, generated on a four-sector instrument using FAB ioni~ation.~' Using a combination of the two approaches to CID it is possible to perform second-generation product ion experiments, in which product ions generated in the expansion region following the sampling orifice are subsequently dissociated further in the collision cell. In this way, the m/z 158 product ion of the MH' ion was shown to be formed via the m/z 576 product ion (generated in the expansion region). Experiments of this type can aid in the understanding of fragmentation pathways and hence the structural elucidation of related compounds. While both means of generating CID provide characteristic fragment ions for increased confidence in analyte identity, the MS/MS mode of operation is more highly specific to the analyte, with CJD occurring after initial mass selection by the first quadrupole. As will be demonstrated later, this feature reduces interferences and greatly improves selectivity, although the increased confidence is gained at the expense of sensitivity.

LCWS of EA Previous reversed-phase LC methods for EA4-' have essentially been modifications of that described orig-

inally by Tsuji and Goetz,' who used a silica-based C18 column and an aqueous methanol/acetonitrile mobile phase with an ammonium acetate modifier. The authors5 documented the effects of mobile phase composition and pH on the retention of EA, and reported no detrimental effects on the EA within the time frame of the LC analysis with a mobile phase pH of 6.2. Despite these assurances, other workers3' recently evaluated resin-based columns, which are applicable over a wide pH range, for the separation of EA and related compounds at pH 8.0 out of concern for their susceptibility to degradation by acid. Unfortunately the chromatographic performance of these columns was significantly inferior to that of the silica-based columns operating at lower pH. In the present investigation a reversed-phase, base deactivated octylsilica stationary phase (Zorbax Rx-C8) was found to give excellent chromatographic performance for the analysis of EA and related compounds, using an aqueous acetonitrile mobile phase acidified with 0.2% formic acid. There appeared to be no significant decomposition of EA on the time-scale of the chromatography, as indicated by good peak shape and linearity of calibration curves (see below). Unfortunately, at the time when this work was done, this stationary phase was available only in a 4.6 mm i.d. format for which the optimal flow rate is around 1 ml min-'. While the ISP interface can be used effectively with full gradient elution. i.e. 100% aqueous to 100% organic, at flow rates of up to 200 pl min-', in our hands optimal performance for robust, quantitative LC/ISP/MS methods, in biological or environmental applications, is obtained at flow rates of the order of 10-50 p1 min-' into the ISP interface. Thus, in the present work the column was coupled to the API source via a postcolumn split arrangement, adjusted such that only 10-15 pl min-' of the 1 ml min-' flow rate was directed to the ISP interface. The result of the LC/ISP/MS analysis of a standard solution of EA (500 ng ml-', 25 pI injected), using selected ion monitoring (SIM) of the protonated molecule (mlz 734) and a linear gradient of 5% to 65% acetonitrile in 15 min, is shown in Fig. 3(a). It should be noted that, while the peak corresponds to an injection oncolumn of 12.5 ng, the split ratio of 100:l implies that the peak represents only 125 pg of EA entering the API source. Although the high post-column split ratio introduced some peak broadening and tailing, the retention characteristics of EA on this column compare favorably with those observed using previous LC rneth~ds,'-'~~' and are entirely adequate for quantitation purposes. This is evident in the LC/ISP/MS calibration curve shown in Fig. 4, generated from serial dilutions of a standard solution of EA over the range 0.01-50 pg m1-l. Duplicate 25 p1 injections were run over an 18 h (overnight) period utilizing the LC autosampler controlled by the mass spectrometer datasystem. The SIM response was linear over the entire range (R' = 0.9998); the data are shown as a log-log plot in Fig. 4 to exhibit the dynamic range, but the linear plot extrapolated through the origin to within experimental uncertainty. Variation of peak areas for replicate injections, over the 18 h period, was less than 2% RSD for all concenrrations used.

ERYTHROMYCIN BY LC/MS

m I

681

1000

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100

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Timc (111 it i ) Figure 3. LC/lSP/MS analyses for EA. Conditions: 250 x 4.6 mm i.d. Zorbax RX-C8 column, mobile phase of aqueous acetonitrile containing 0.2% formic acid at a flow rate of 1 ml min-’ (split 1 : 100 to mass spectrometer), linear gradient from 5% to 65% acetonitrile in 15 min, 25 pl injection; SIM of the protonated molecule (m/z 734.4). (a) Standard solution (500 ng m l - l ) of EA (i.e. 12.5 ng injected on-column, and thus 125 pg delivered to the mass spectrometer). (b) Control salmon extract spiked with EA at a level equivalent to 0.1 pg g - ’ (0.1 ppm) of flesh extracted; 25 pl injected from an extract 5 g ml-’ tissue equivalent (i.e. 12.5 ng injected on-column assuming 100%recovery). (c) Control salmon extract spiked with EA at a level equivalent to 0.01 pg g-’ (0.01 ppm) of flesh extracted; 25 pI injected from an extract 5 g ml-’ tissue equivalent (i.e. 1.25 ng injected on-column assuming 100% recovery). (d) Control salmon tissue extract.

Analysis of EA in spiked salmon tissue In order to evaluate the applicablity of this LC/MS methodology to real-world fish flesh samples, a preliminary extraction procedure had to be adopted. Takatsuki et d”+” developed an efficient method for the GC/MS analysis of EA in beef and pork based on an initial methanol extraction followed by a defatting of the extract with hexane and a partitioning between chloroform and a phosphate buffer. It has been reported, however, that acetonitrile can provide a more selective extraction of antibiotics from tissues with a high lipid content, such as that of salmonids.” Indeed, acetonitrile has been used previously for extraction of EA from animal tissues, milk and egg.3 Therefore, initial experiments compared the use of methanol and acetonitrile for the initial extraction, followed by Takatsuki’s liquid-liquid partitioning clean-up with only minor modifications such as substitution of methylene chloride for chloroform (for toxicity reasons). As reported by other worker^,'-^,^*^' it was found that EA is unstable in slightly acidic and neutral media.

Figure 4. Calibration curve, shown as a log-log plot to display the dynamic range, obtained for standard solutions of EA (duplicate 25 pI injections) by LC/ISP/MS using SIM (m/z 734, MH+). Conditions as in Fig. 3, except that all injections were performed by an unattended autoinjector. The points are averages of duplicate measurements. The line represents a linear regression fit ( R 2 = 0.9998) to the data.

It is important therefore that any partitioning with aqueous solutions be carried out quickly to prevent degradation. For the experiments reported here, after removal of the methylene chloride on a rotary evaporator, the extract from 25 g tissue was reconstituted in 5 ml of methanol, and 25 p1 aliquots were injected directly on to the reversed-phase LC column (see Experimental). Both extracting solvents provided good recovery of EA (>90% at the 1 ppm spike level), but the acetonitrile extracts were cleaner, as indicated by color and turbidity of the final extract, and by the presence of many additional peaks in the LC/MS total ion current (TIC) chromatogram of the methanol extract. Therefore, acetonitrile was selected for all subsequent work. Although the extraction yields proved adequate, this present extraction procedure does not provide sufficient cleanup. Deterioration of the LC column efficiency was apparent after a number of injections of extract. This column deterioration was accompanied by a conversion of EA to AEA at the head of the column, presumably due to a build-up of co-extractives. It was necessary to use a guard column and to change it frequently. Since it is clear that this extraction procedure is not entirely satisfactory, an improved clean-up procedure is currently under development and will be reported separately. Although there is currently no specified tolerance level set for residual EA in cultivated fish in Canada, there are maximum residue limits set for the antiobiotic in food products from terrestrial livestock. These include 0.1 and 0.125 ppm (pg g-’) in the edible tissue of swine and chickens, r e ~ p e c t i v e l yIn . ~ ~order to evaluate the present LC/ISP/MS methodology, control salmon tissue was spiked with EA at levels between 0.01 and 1 ppm. Spiked tissue was extracted and cleaned up as described above, and analyzed by LC/ISP/MS using a variety of scan modes. The TIC trace obtained from the full-scan LC/ISP/ MS analysis of an extract of control salmon tissue

S. PLEASANCE ET A L

!

!I

1 '.,"

-

s

EA

I

0.0

2.0

4.0

6.0

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8.0 10.0 12.0 14.0

Time (min) Figure 5. LC/ISP/MS analysis of an extract of control salmon tissue spiked with EA at a level corresponding to 1 pg of EA per gram of tissue extracted (1 pprn). Conditions as in Fig. 3, except a mass range 100-800 Da with a dwell time of 5 ms per dalton. (a) Full-scan TIC; (b) reconstructed ion chromatogram for m/z 734.4 (MH+ for EA).

spiked with EA at the 1 ppm level is shown in Fig. 5(a). Several early-eluting peaks are observed in the TIC trace, and these are assigned to endogenous compounds by comparison with those obtained for non-spiked tissue extracts. At this level of fortification only a very weak response is observed in the TIC trace at the correct retention time for EA. The reconstructed ion chromatogram for m/z 734.4 (Fig. 5(b)), however, reveals a strong signal for EA, well resolved from any interferences. The background-subtracted mass spectrum recorded at the crest of this peak (not shown) was dominated by the MH' ion. Recognizable spectra in which the MH' ion at m/z 734.4 remained the base peak were obtained for similar extracts of control salmon flesh spiked at the 0.5 ppm level. Using a more limited mass range (400-750 Da), confirmatory mass spectra could be obtained at even lower levels, but at and below the probable regulatory limits of 0.1 ppm the full-scan approach was not sufficiently sensitive. The sensitivity of the LC/ISP/MS method can be readily increased through the use of SIM techniques. The SIM chromatogram of a control tissue extract, under LC conditions identical to those described thus far, is shown in Figure 3(d). Apart from a large peak eluting close to the solvent front, there were no potential interferences from the matrix close to the retention time for EA standards (Fig. 3(a)). The corresponding

SIM chromatogram of an extract of tissue spiked with EA at the 0.1 p.p.m. level is shown in Figure 3(b). The presence of EA is easily confirmed at this level with excellent signal-to-noise ratio. Figure 3(c) shows the response obtained for the equivalent of a 0.01 ppin (10 ppb) spike (25 pl injection, corresponding to 1.25 ng injected on-column assuming 100% recovery). The observed signal-to-noise ratio of about 20 : 1 suggests that the method detection limit is probably about 1 ppb in the present state of development; recall, however, that the level of co-extractives was such as to seriously limit column life. An improved extraction/clean-up procedure will be necessary, and with additional preconcentration should be capable of providing even lower detection limits. However, it is already clear from the present work that the LC/MS approach will provide more than adequate sensitivity, provided that regulatory levels for EA in salmon flesh are set at values comparable to those currently established3j for terrestrial livestock. Unfortunately the combined specificity of a reproducible retention time, well resolved from endogenous compounds, plus a high-mass MH' ion well separated from chemical noise, is not in itself considered to be sufficient for the confirmation of analyte identity for legal purposes. Stemming from criteria established when GC/MS using EI was the only viable analytical approach for many regulatory laboratories, it is generally considered necessary to monitor at least three ions for confirmatory analyses.34 In the present case it is possible to confirm the presence of EA using at least three ions (m/z 734.4, 576.3 and 158.1, Fig. 2), for extracts of salmon flesh spiked at the putative regulatory level (0.1 ppm) with a 25 pl injection, by applying the conditions established earlier for generating CID in the API source, i.e. at higher orifice voltages. In order to fully utilize the power of the triple-quadrupole instrument, however, one can use conventional MS/MS techniques in which mass selection occurs both prior to and after CID in the collision cell. This approach is illustrated in Fig. 6, which shows the LC/ISP/MSiMS analysis of a salmon flesh extract spiked at the 50 ppb level, using multiple reaction monitoring (MRM). Good responses were observed for all three M H + dissociations monitored, thus significantly increasing the degree of confidence which can be placed in the analysis even for levels less than 0.1 ppm. An extensive study of spike recoveries was not undertaken in the present work. However, based upon the few experiments of this kind conducted using both salmon and trout flesh homogenates, the indicated recoveries fell in the range 107 & 18%. A complete evaluation of recoveries will be reported separately as part of the improved clean-up procedure currently under development. Identification of EA and its metabolites in flesh of salmon fed on medicated feed One of the objectives of this preliminary investigation was to develop a robust LC/MS method for the determination of EA, with the long-term intent that the method could be used to establish safe withdrawal

ERYTHROMYCIN BY LC/MS

a)

683

m/z 734 > 158

il

I 10.0

I

1,

-

i.I!

12.0

* ~

A

U

L

.,

*/

> I _ _

0.0 2.0 4.0 6.0 8.0 10.0 12.0 14.0

II 14.0

450 500 550 600 650 700 750 800 850 m/z

c) 10.0

12.0

c) m/z734>576

1410

I

1

7 16.4

100,

l

I

558.3

540 3

i

LA&.--____

450 500 550 600 650 700 750 800 850 11liL

A I

10.0

12.0

14.0

Time (min) Fig. 6. LC/ISP/MS/MS analysis, using selected reaction monitoring, of an extract of salmon tissue spiked with EA at the 0.1 ppm level. Reactions monitored were the dissociations of the protonated molecule (m/z 734) to (a) m/z 158, (b) m/z 558 and (c) m/z 576 (all m/z values truncated). LC conditions were the same as those used to obtain Fig. 3; MS/MS conditions were as in Fig. 2(c), except using a dwell time of 200 ms per reaction.

periods for EA in cultured salmon following administration of medicated feed. It is necessary therefore to identify metabolites of EA, which are likely to be included in any future regulatory protocol. The present approach was therefore applied to the analysis of fish that had been exposed to EA through a controlled laboratory feeding experiment. Fish slaughtered after 9 days of medication were used. As expected, analysis of the tissue extract by full-scan LC/ISP/MS (not shown) indicated a very high level of EA in the tissue (43 ppm, uncorrected for recovery efficiency). In addition, the presence of several other components eluting close to EA was noted. On the assumption that these components were related to EA, the extract was re-analyzed with the mass spectrometer configured for a precursor ion scan mode tuned to the m/z 158 product ion (protonated dehydrodesosamine) of EA. Some results of

Figure. 7. LC/ISP/MS/MS analysis of salmon tissue extract from a feeding experiment. A precursor ion scan mode was used, for a selected product ion m/z 158. LC and collision conditions as in Fig. 6, except that a mass range of 400-800 Da with a dwell of 8 rns per dalton was used. (a) Reconstructed TIC, all precursors of m/z 158 in the range m/z 400-800; (b) precursor spectrum acquired at crest of peak 1 (confirmed to be EA) in Fig. 7(a); (c) precursor spectrum acquired at crest of peak 2 (confirmed to be AEA) in Fig. 7(a).

this LC/ISP/MS/MS analysis are summarized in Fig. 7. For convenience in the discussion of these and other data. Table 2 lists the LC/MS peak numbers together with retention times, m/z values of the MH' ions (and of any fragment ions observed), as well as the identifications of the compounds. The TIC (Fig. 7(a)) suggests the presence of at least five components which gave rise to the product ion at m/z 158, in addition to EA itself (peak 1). The precursor ion tandem mass spectrum of EA, given in Fig. 7(b), shows three ions at mlz 558, 576 and 734 which correspond to those ions observed in the product ion spectra of EA (Fig. 2(c)). The most intense peak in the TIC (peak 2) gave the precursor ion spectrum shown in Fig. 7(c). This spectrum reveals three precursors of m/z 158 at m/z 540, 558 and 716, each 18 Da lower than the corresponding ions observed for EA. On this basis, peak 2 could possibly be assigned to anhydroerythromycin (AEA), EA enol-ether (EAEN), or pseudoerythromycin A enol-ether (psEAEN) (see Fig. 1 and Table 1). A standard sample of AEA, a known acid degradation

S . PLEASANCE ET AL.

684

Table 2. Summary of LC/MS and LC/MS/MS data for extract of flesh of salmon fed on medicated feed. Identities given are tentative for those marked with a query; compounds marked with an asterisk are those for which an authentic standard was available Peak no.

1

2 3 4 5 6 7 8 9

10 11 12

Ref time (min)

11.3 12.3 11.1 11.6 12.0 12.7 11.4 13.0 10.6 11.1 12.4 10.2

MH'

(rn/z)

734.4 716.4 702.4 702.4 702.4 702.4 71 6.4 71 6.4 720.4 720.4 720.4 750.4

product of EA, gave an identical retention time and mass spectrum to that of peak 2 in Fig. 7(a), supporting its identification as AEA. Similar precursor spectra (not shown), obtained at other retention times (Fig. 7(a)), gave evidence for MH+ ions at m/z 750,720 and 702. Protonated molecules MH' at m/z 702, 716, 720, 734 and 750, thus detected by LC/ISP/MS/MS as precursors of the fragment ion at m/z 158, were then included in an LC/MS SIM experiment. The SIM mass chromatograms thus obtained are presented in Fig. 8. The individual components are now more clearly defined than in the comparable TIC trace (Fig. ?(a)), with excellent resolution and signal-to-noise ratios. At least four peaks (peaks 3-6) are observed in the chromatogram for m/z 702 (Fig. 8(a)); possible metabolites, degradation products or trace contaminants at this m/z include anhydroerythromycin C (AEC, N-demethylerythromycin A enol ether (dMeEAEN), anhydro-N-demethylerythromycin A (AdMeEA) and N-demethylpseudoerythromycin A enol ether (dMepsEAEN) (Fig. 1 and Table 1). Only AEC was available as a standard to test these proposals, and its retention time did match with that of peak 4;note that this compound, like EC itself, is unlikely to have been formed as a metabolite or decomposition product of EA, and was therefore more likely to have been present as an impurity in the medicated feed (see later). The major peak in the m/z 716 chromatogram (peak 2) wtrs confirmed to be due to AEA (Fig. 1 and Table 1) via comparison with an authentic standard, as discussed above. The minor peak (peak 8) eluting after AEA was shown to correspond to EAEN, again by comparison with a synthesized standard; the trace peak (peak 7) eluting before AEA possibly corresponds to psEAEN (Fig. 1 and Table l), but no standard was available to test this speculation. The three peaks in the m/z 720 mass chromatogram (Fig. 8(c)) all represent demethyl forms of EA, and the major peak (peak 10) presumably corresponds to the well-known'.2*28N-demethyl metabolite of EA (dMeEA, Fig. 1 and Table 1). Other known isomers which could account for the two minor peaks (peaks 9 and 11) are erythromycin C (EC) and dMepsEAHK (Fig. 1 and Table 1); an authentic standard of EC gave a retention time identical to that of peak 9. Only one peak (peak 1) is apparent in the SIM

Fragment ions (rn/z)

Identity

716, 576, 558, 158 558,540,158 544 558, 540, 522, 158 544, 526, 508, 144 544, 526,144

EA* AEA* d MepsEAEN ? AEC* Ad M e EA ? dMeEAEN? ps EAEN ? EAEN? EC* dMeEA? dMepsEAHK? ?

558, 540,158 576, 558, 158 702,562,544, 144

chromatogram for m/z 734 (Fig. 8(d)), at a retention time corresponding to that of EA itself; this observation suggests that the ring contraction of the macrocyclic lactone (C13 -+ C11 translactonization), shown'.2 to lead to the pseudoerythromycin A series, did not occur to any significant extent under the conditions of the present work. At least five peaks were observed in the SIM chromatogram for m/z 750 (Fig. 8(e)). A possible metabolite with the appropriate molecular weight is EANO, the N-oxide of EA (Fig. 1 and Table 1); comparison with a synthetic standard showed that the most intense peak 12 does not correspond to EANO. This result was unexpected, since the product ion spectrum of m/z 750 for peak 12 (Table 2) is entirely consistent with an M H + precursor which readily expels a neutral fragment of 16 Da, to yield a fragment ion at m/z 734 which in turn fragments further in a fashion indistinguishable from that of protonated EA; however, experience with other protonated N-oxides has shown that they expel a water molecule, rather an oxygen atom, under low-energy MS/MS conditions. In fact the retention time of the EANO standard matched that of peak 13 in Fig. 8(e), and peak 12 remains unidentified. Erythromycin F (EF, Fig. 1 and Table 1) has the same molecular weight (MH' at m/z 750), but was not detected as an impurity in a similar LC/MS analysis of the medicated feed (see below) and is unlikely to be formed as a metabolite or degradation product of EA. Similarly, erythromycins B and D (Fig. 1 and Table 1) were not detected in either the feed or the salmon. In the absence of standards other than EA, AEA, EANO, EAEN, EC and AEC, supporting evidence for the tentative assignments could be obtained only by additional LC/ISP/MS/MS analyses using product ion scans (the amounts available were far too small for characterization by NMR to be feasible). While these product ion spectra (see below) were consistent with the present assignments, they do not provide conclusive evidence of identity. Using the acquisition software package provided by the manufacturer of the mass spectrometer, it was possible to set up the instrument for an LC/MS/MS experiment in which precursor ions and associated scan ranges were changed at appropriate times during the elution profile, and thus to obtain

685

ERYTHROMYCIN BY LC/MS

702

544

701

C)l00l

I

I 558

702

e)ioo,

i

131

100

200

300

400

500

600

700

!lI/7

Figure 9. LC/ISP/MS/MS analysis of tissue extract of salmon from a feeding experiment using product ion scan mode. Conditions as in Fig. 7 except that precursors of m/z 702.4 were selected, and a product ion m/z range of 100-71 0 was scanned with a dwell time of 5 ms per dalton. (a) Reconstructed TIC (m/z 702.4 --t 1100, 7101) ; (b) product ion spectrum of m/z 702.4, peak 3;(c) product ion spectrum of m/z 702.4, peak 4;(d) product ion spectrum of m/z 702.4, peak 5; (e) product ion spectrum of m/z 702.4, peak 6.All annotated m/z values are truncated, not rounded

C

0.0

5.0

10.0 15.0 Time (min)

20.0

Figure 8. LC/ISP/MS analysis of a tissue extract of salmon from a feeding experiment, using SIM (all annotated m/z values are truncated). Conditions as in Fig. 3 except with a dwell time of 100 ms per SIM channel. Relative intensity scales: (a) 16; (b) 100; (c) 28; (d) 40; (e) 4.

several product ion spectra of mass-selected precursors in a single run. These LC/MS/MS runs were performed unattended using the LC autosampler. Partial results of one such set of experiments are summarized in Fig. 9; the LC/MS/MS data shown are for M H + precursors at m/z 702.4, with fragment spectra scanned over the range m/z 100 to 710. The reconstructed TIC from this experiment (Fig. 9(a)) agrees well

off.

with the SIM chromatogram for the same extract solution shown in Fig. 8(a). The product ion spectra of m/z 702.4, corresponding to peaks 3-6 (Fig. 8(a)), are shown in Fig. 9(b)-(e). Identification of peak 4 as AEC was straightforward since a standard was available ;the fragment spectrum is dominated by a neutral loss of 144 Da rather than the 158 Da characteristic of the cladinose moiety, confirming that the neutral sugar is demethylcladinose in this case, while the fragment ion at m/z 158 is characteristic of the N-dimethyl amino-sugar desosamine. Of the other three possibilities with MH' ions at m/z 702.4, all are N-demethyl compounds (dMeEAEN, AdMeEA and dMepsEAEN) and are predicted to give an amino-sugar residue ion at m/z 144 rather than 158, and a neutral loss of 158 Da corresponding to the cla-

686

S. PLEASANCE ET A L

dinose moiety. Each of peaks 5 and 6 satisfies both of these requirements (Fig. 9(d) and 9(e)), and the lowintensity peak 3 may also, although the low-mass fragment was not observed in this case (Fig. 9(b)) possibly due to the low intensity available. Peak 3 is assigned tentatively to dMepsEAEN, principally because of its low intensity as predicted for the unusual macrolide structure e (Fig. 1). There is no firm information available on which of peaks 5 and 6 should be assigned to which of AdMeEA and dMeEAEN; the assignments within this pair, given in Table 2, are therefore uncertain and may be reversed, though it may be noted (Table 2) that AEA elutes before EAEN. Similarly, of the three peaks in the SIM trace for m / z 720.4 (Fig. 8(c)), peak 9 is confidently assigned to EC (standard available) while the intense peak 10 is probably dMeEA since N-demethylation appears to be a favoured metabolic pathway; the low-intensity peak l l is then tentatively assigned to dMepsEAHK as the only other candidate in Table 1. It is important to note that most of these related compounds (Figs 7-9, Table 2), identified or tentatively identified in the present work, are degradation products or metabolites of EA in the fish flesh. The only compounds listed in Table 1, which were detected in the feed extracts, were EA itself plus AEA, EC and AEC; the latter two compounds were present at levels < 1%. It is of interest that the EC and AEC compounds appeared to accumulate in the fish relative to EA plus AEA, and that both of these anhydro compounds appeared in the fish flesh at abundances greater than in the feed, relative to their respective parent compounds.

labile molecules in complex biological matrices. The instrumental sensitivity has been demonstrated to be more than adequate for detection and confirmation of EA in salmon flesh, if regulatory levels are set at values similar to those currently in effect for pork and chicken.33 However, during the course of the present study it was realized that, if the methodology is to be applicable to high-throughput quantitative analyses. much more effective clean-up of the samples will be required to remove the large quantities of coextractives. The procedure here was acceptable for a few sample injections, after which the guard column had to be replaced. An effective clean-up procedure is currently under development, and will be reported separately together with results on recovery efficiencies, improved detection limits, and dynamic range for the procedure as a whole. All existing analytical methods for EA, including that described in the present work, suffer from the lack of a suitable surrogate internal standard and this is also an objective of the extended research. Volume control internal standards are less important for LC (injector loop technology) than for G C , but do provide protection against errors arising from uncontrolled solvent evaporation and should be easier to devise. The present work has also demonstrated the great diagnostic capabilities of the LC/MS/MS approach, in both precursor ion and product ion modes. The utility of this approach has been demonstrated here both for confirmation of analyte identity and for partial structural characterization of unknown analytes.

Acknowledgements

CONCLUSION

This work was funded in part by the Bureau of Veterinary Drugs, Health and Welfare Canada. The authors are grateful to Mrs Pearl Blav I I M B I and Ms Jacaualine Walisser (UBCI for technical assist-

power of ionspray LC/MS in the analysis of polar,

NMRanalyses.

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