Anal. Chem. IQQI, 63,1709-1794

1789

Electrospray Ionization Mass Spectrometry of Semduramicin and Other Polyether Ionophores Richard P. Schneider, Martin J. Lynch, Jon F. Ericson, and Hassan G. Fouda*

Drug Metabolism Department, Central Research Division, Pfiter Znc., Groton, Connecticut 06340

Pneumatically assisted electrospray mass spectrometry of poiyether ionophores yldds several molecular bns. A 8metal adduct molecular ion can be obtained by the addltlon of a neutral salt to the HPLC mobile phase. This approach may be useful in rbuctuai studks d unknown knophorm and In the development of specific methods for their analysis in complex matrices. Cdildon-induced dksociaiion of the molecular ions provides addltional structural Information and enhanced tqmclflctty for trace analysis. HPLC mobllaphase compodtion and flow rates have been optimized for on-line analysis. Bed response and lowed background nobe were obtained at the flow rate of 40 pL/mln of a moblle phase containing a 20180 mlxture of water and acetonltrile. The development of a speclfic confirmatory assay for the new ionophore semduramicin In chlcken liver demonstrates the usefulness of on-line HPLC pneumatically assisted electrospray mass spectrometry.

INTRODUCTION Polyether ionophores, also called polyether antibiotics, are fermentation-derived biologically active compounds characterized by the presence of a carboxylic acid group and several cyclic ether units (Figure 1). The term ionophore refers to their ability to form stable complexes with alkaline cations. Complex formation is achieved by surrounding the cation with the oxygen functions at the center of the molecule, leaving the lipophilic alkyl groups on the outer surface of the complex. The ionophores have been classified on the basis of their cation selectivity (monovalent or divalent) and their chemical structure ( I ) . They exert their biological activity by catalyzing an electroneutral cation proton exchange across cell membranes, moving as undissociated acids in one direction and as neutral complexes in the other (2). Their usefulness as cardiovascular drugs has been evaluated (3, 41, but their commercial success is due to their wide utilization as anticoccidiosis agents in broiler chickens (5) and as feed efficiency enhancers in cattle and sheep (6). Semduramicin is a newly discovered carboxylic acid ionophore for the management of chicken coccidiosis (7). An HPLC spectrophotometric method has been developed for the routine monitoring of semduramicin residues in edible chicken meats (8). A much more specific confirmatory assay is required by regulatory authorities (9). In most cases, the stringent specificity requirement can only be met by mass spectrometry. Mass spectrometry of carboxylic acid ionophores has recently been reviewed (10). Electron ionization provided structural information and has generally featured molecular ions of very low abundances. Additional structural infor-

*Towhom corres ondence should be addressed: Principle Research Investigator, b r u Metabolism, Central Research Division, Pfizer Inc., Groton, CT b340. Telephone 203-441-4647. Fax 203441-4109. 0003-2700/91/0383-1789%02.50/0

mation and more abundant molecular ions were obtained by negative-ion electron ionization (11). Chemical ionization (12, 13)and various desorption ionization techniques in both the p i t i v e and negative modes have been described (13,14). The application of fast atom bombardment mass spectrometry to various classes of carboxylic ionophores has also been reported (15). In all classes, protonated molecular ions were absent but metal adduct molecular ions were evident. Desorption chemical ionization (DCI) and on-line thermospray HPLC mass spectrometry have been compared for the confirmation of one ionophore in chicken tissues. The authors (16) stated that the first approach was totally inadequate and the second was considered marginally acceptable (16).Only thermospray tandem mass spectrometry generated reproducible data for a confirmatory method (16). Electrospray ionization (17) has emerged in recent years as one of the most broadly applicable soft ionization techniques. Ion spray (IS), also called pneumatically assisted electrospray (18),utilizes electrostatic and aerodynamic forces that accommodate liquid flow rates higher than those of pure electrospray. This facilitates the on-line coupling of HPLC and mass spectrometry. Current interest in electrospray is, for the most part, due to the formation of multiply charged ions which extended mass spectrometric analysis to peptides and other macromolecules (19,20). However, the high ionization efficiency of this technique has also allowed the generation of impressive results for small molecules (18, 21). In this report, we describe the application of IS to the HPLC/mass spectrometric analysis of polyether ionophores. The low detection limits and enhanced selectivity of this approach are illustrated by the development of a confirmatory assay for the new ionophore semduramicin in chicken liver.

EXPERIMENTAL SECTION IIPLC/Mass Spectrometry. An AB1 140A dual-syringe pump was used to deliver the mobile phase at a constant rate. Sample injections were made with a Rheodyne 7413 microbore injection valve containing a 1-pL internal loop. In order to optimize conditions for ionophore analysis, changes in eluent flow rate and composition were evaluated by flow injection analysis. The HPLC flow rate was varied from 5 to 100 gL/min. The mobile-phase composition was a binary mixture of organic solvent and water. The organic portion was acetonitrile/tetrahydrofuran (85/15) and was varied from 10% to 90%. For chromatographic analysis, the aqueous/organic (10/90) mobile phase was delivered at 40 pL/min. The aqueous portion was 5 mM sodium acetate, and the organic portion was CH,CN/THF (85/15). The analyticalcolumn was a Hyperd BDS (CIS)microbore column (1X 250 mm) preceded by a microbore in-line filter (0.5 pm). The column effluent was interfaced to the ionspray probe by -6 in. of fused-silica tubing (100 pm i.d.). Mass spectral analysis was performed on a SCIEX API I11 triple-quadrupole HPLC/MS/MS system. The mass spectrometer was equipped with an ion spray (IS) interface set at a voltage of 3900 V and a nebulizer gas pressure (nitrogen)of 22 psi. The nitrogen curtain gas was adjusted to a constant flow rate of 1.2 L/min. Positive ions formed at atmospheric pressure were sampled into the quadrupole mass filter via a 0.0045-in. pinhole aperture. For CID experiments, the collision energy was -48 V for the laboratory frame and the collision gas thickness was set @ 1991 American Chemical Society

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ANALYTICAL CHEMISTRY, VOL. 63,NO. 17, SEPTEMBER 1, 1991

Table I. Molecular Ions Prellent in Electrollpray Spectra of Several Ionophores Following the Addition of a Neutral Salt (5 mmol) to the Mobile Phase"

ionophore

molwt

maduramycin lasalocid salinomycin semduramicin

916 590 750 872

monoisotopic molecular ions (mlz)detected following the addition of a specific salt nosalt NaCl KCl CsCl 6

608' 773d 89W

939 613 773

895

955 629 789 911

1049 723 883 1005

"The mobile phase for these experiments was a 72/13/15 mixture of acetonitrile/THF/watr. The quadrupole mass spectrometer was set for a maas accuracy of 0.1 m u . bIonization of the analyte is suppressed in the absence of salts. See text. Cother low-abundancemetal adducts were also present. dThe sodium salt of salinomycin was used.

Flgwe 1. Molecular structures of semduramlcin and several selected

and normal (silica gel) phase extraction columns. Typically, a 1.25-g liver sample was weighed into a disposable borosilicate culture tube, vortexed for 3 min in 7.5 mL of 8/2 methanol/l% NH,OH in water, incubated for 1 h at 55 OC, and centrifuged. The supernatant was concentrated under nitrogen at 55 "C to an approximate final volume of 2-3 mL. A 5-mL aliquot of water was added, and the solution was vortexed and applied to a preconditioned C8BondElute solid-phase extraction column, washed with 3 mL of water, followed by 1mL of 25/75 methanol/water. The BondElute column was previously prepared washing with 5 mL aliquota of acetonitrile, methanol, and distilled water, respectively. Semduramicin was eluted with 5 mL of ethyl acetate into a disposable culture tube, and the eluate was dried under nitrogen at 50 "C. The residues were reconstituted with 6 mL of 1/1methylene chloride/isooctane, briefly vortexed, and sonicated for 5 min. The reconstituted sample and l mL of 1/1 methylene chloride/isooctane rinse were poured directly into a silica BondElute column. The silica BondElute column was previously preconditioned with approximately 5 mL of chloroform followed by 5 mL of 1/1 methylene chloride/isooctane. The BondElute column was washed with 2.5 mL of 1/1 methylene chloride/isooctane followed by 1mL of ethyl acetate. Semduramicin was eluted with 5 mL of 18/2 methylene chloride/ methanol into a disposable culture tube and dried under nitrogen at 50 OC. The residue was dissolved in 50 pL of mobile phase, and 5-pL aliquota were injected for LC/MS/MS analysis.

at 7.6 X 1014atoms/cm2. For multiple-reaction monitoring of semduramicin, the mass spectrometer was adjusted to selectivity monitor parent ion to daughter ion fragments of m / z 895.5-852 and also m/z 895.5-834. Voltage parameters of the quadrupoles and Brubaker lenses used during sample analysis were as follows: OR 65, RO = 30, R1 = 26, L7 = -36, R2 -18, R3 = -44, L9 = 15. Voltages for the Faraday plate and the multiplier were 145 and -4000 V, respectively. The channel electron multiplier was operated in a pulse-counting mode and was capable of recording 4 X 10s counts/s. Accurate mass measurement of certain metal adduct molecular ions was obtained by flow injection analysis (FIA) using a 5-pL loop and a flow rate of 20 ctL/min. The mass spectrometer was set for a mass accuracy of 0.1 amu. Otherwise during HPLC/MS/MS analysis, the mass accuracy was set at 0.5 amu (RE = 120). Materials. Stock solutions (2 pg/mL) of salinomycin, maduramicin, lasalocid, and semduramicin were prepared in acetonitrile/water (9/1) and stored under refrigeration. Dilutions to the desired concentrations ( 4 0 0 ng/mL) were made prior to use. Aqueous chloride salts, NaCl, KCl, or CsCl, were used to enrich ionophore solutions to a final concentration of approximately 5 mM. Solvents used for extractionsand chromatography were HPLC grade reagents (Baker). BondElute columns and the VAC ELUTE manifold were purchased from American Bioanalytical. Extraction of Semduramicin from Chicken Liver. Semduramicin was extracted from liver with 8/2 methanol/l% NH,OH in water and separated from coextractives on reverse (Cd

RESULTS AND DISCUSSION Formation of Metal Adducts. Initial attempts to analyze a carboxylic ionophore, semduramicin, by IS resulted in several low-intensity molecular ions, including an ammonium adduct (m/z 890),a sodium adduct ( m / z 895), and other ions. The formation of several molecular ions complicates structural analysis and would dilute the ion beam. We have investigated shifting the molecular ions to a specific metal adduct by the selective addition of one salt to the mobile phase. The results shown in Table I indicate the formation of a single metal adduct molecular ion (I + Met)+by the addition of an alkaline metal (Met) to the ionophore (I). Higher ionization efficiency was also evident after the addition of a salt. For example, instrumental response for 100 ng of lasalocid increased 75-fold after the addition of sodium chloride to a saltless mobile phase. Different ionophores have different affinity for group I and I1 cations, depending on their classification (Le. monovalent or divalent polyethers). The divalent glycoside, lasalocid, is known to form dimers with univalent metal ions (5,22). In the presence of sodium chloride, its IS spectrum (Figure 2) showed an abundant sodium adduct molecular ion (I + Na)+ at m / z 613 and a much less abundant dimer (21 + 2Na - 1)+ at m / z 1225. Dimerization of lasalocid was only evident in the presence of a sodium salt. No dimers were observed for the monovalent ionophores salinomycin, maduramicin, or semduramicin. However, complexation of one ionophore

CY O A O H

MADURAMlClN

SAUNOMVCIN

OH

CY

LASALOCID

ionophores.

ANALYTICAL CHEMISTRY, VOL. 63, NO. 17, SEPTEMBER 1, 1991 11 + Na]

+

100.

[M

'"1

613.6

4

1791

+ Na-C02-H20]+ 834

614.5

75.

50.

11 +NH,]+ 608.5

25.

E *

[M + Na-C02] +

i

a

[M + Na] +

852 895

550

750

650

850

950

MI2

Figwe 4. CID daughter spectrum of semduramicin sodium, mlz 895. [M + N~-COI-HIO] 877

loo) MR

Flgurr 2. Ion spray mass spectrum of lasakcid-sodium, showing the accurate mass (0.1 amu) of [I NH,]', [I Na]' and [21 + 2Na

-

+

1]+.

[I + CS]

+

g

>

t fn z w

!-

1

+

75

I

I

+

I I

50

w

z2

'

[M + Na-C02] 895

25.

0 355

I

435

515

595

675 MI2

755

835

[M+Na]+

939

I l l915, I

I

995

Flgurs 5. CID daughter spectrum of maduramicin sodium, mlz 939.

MI2

Figure 3. Ion spray mass spectra of salinomycln in the presence of cesium chloride, showing the accurate mass (0.1 amu) of [I Cs]+

and [I

+ 2Ce - I]+.

+

molecule with two cations was evident in both the monovalent and divalent ionophores (Figure 3). The selective formation of metal adduct molecular ions by doping the mobile phase with salts may aid molecular weight determination of unknown ionophores, the investigation of their metal ion preference and their propensity for dimer formation. At present, the metal ion preference and dimer-

ization data are generated from X-ray crystallography (23,24) and NMFt studies in nonpolar solvents (25).It could be argued that data derived from the electrospray mass spectrometry of aqueous solutions may be more relevant to biological systems. The formation of metal adduct molecular ions can also be useful in analytical studies. Allowing the selected-ion monitoring (SIM) of any of several possible molecular ions can render analytical methods more specific and can easily avoid interferences from matrices. For example, selected-ion monitoring of salinomycin residues can be based on any of the following ions: m/z 773 (I + Na)+, m / z 789 (I + K)+,or m / z 883 (I + Cs)+, depending on the salt added to the mobile phase. Collision-Induced Dissociation (CID) Spectra. The CID daughter-ion spectra of semduramicin and maduramicin are shown in Figures 4 and 5, respectively. In both cases,the sodium adduct molecular ions are dissociated to two fragments. The minor daughter ion is consistent with the neutral loss of a COPmolecule from the molecular ion. The most abundant daughter ion resulted from the loss of 62 Da. A similar loss was reported to occur in ionophores having a 8-hemiketalcarboxylic acid by fast atom bombardment (Siegal et al. (15)). That loss is reportedly initiated by an intramolecular proton transfer from the carboxylic acid to the hydroxy group of the 8-hemiketaland then followed by the concerted losses of water and carbon dioxide to produce a polyether olefin (Siegal et al. (15)). Molecular orbital calculations indicated that the concerted loss of both C02 and HzO is much

ANALYTICAL CHEMISTRY, VOL. 63, NO. 17, SEPTEMBER 1, 1991

1792

s

600K

>

I-

t z W

I

u)

PW

I

c

L

5

8o K1

W

501

I

400K

Y

4n

a 200K

1225 0 395

500

605

710

815 MIZ

920

1025

1130

1235

oi

Flgure 6. CID daughter spectrum of lasalocid sodium dimer, m l z 40K-

1

0

I

I

20

40

I

80

h

background -2K

dz

I

P

I

0

v)

9 W

sm

Y

2 20K-

-lK

'

1

100

Yo ACETONiTRiLE IN THE MOBILE PHASE Figure 8. Effect of mobile-phase composition on the selected-ionmonitoring analysis of saiinomycin at mlz 773.

-3K

30K-

I

1

60

5