Pharmaceutical analysis using fast atom bombardment mass spectrometry-mass spectrometry

178

Combined use of liquid and gas chromatography An alternative approach was considered to be attractive, 1 e utilization of hqurd chromatography for class fractionation and collectron of fractions with their subsequent gas chromatographrc analysis for quantitative evaluation9 The cornpositron of the ortgmal gasoline sample 1s calculated from the GC results, utihzmg the close uniformity of the FID response for hydrocarbons The most serious shortcomings of the method are related to the mterference of the mobile liquid phase by GC analysis It is very important to know exactly the volume of the collected fractions The accuracy of the method of calculatton depends srgmficantly on the exact reproducrbihty of the ahquot volumes mlected mto the gas chromatograph References 1 R L Miller, L S Ettre and N G Johansen, J Chromatogr , 259 (1983) 393 2 D 1319-77, Hydrocarbon Types m Ltquld Petroleum Products by Fluorescent Indicator Adsorption, American Society for Testing and Materials,, Philadelphia, PA, 1981 Book of ASTM Standards, Part23, pp 703-713

trendsVIanalyticalchemutry, vol 4, no 7,1985

‘

3 I M Whlttemore, m K H Altgelt and T H Gouw (Edltors), Chromatography m Petroleum Analysis, Marcel Dekker, New York, 1979, pp 50-70 4 D H Desty, A Goldup and W T Swanton, in N Brenner, J E Callen and M D Wels (Editors), Gas Chromatography, Academic Press, New York, 1962, pp 105-138 5 W N Sanders and J B Maynard, Anal Chem , 40 (1968) 527 6 E R Adlard, A W Bowen and D G Salmon, J Chromatogr , 186 (1979) 207 7 N G Johansen, L S Ettre and R L Mdler, J Chromatogr , 256 (1983) 393 8 S HAla, M KuraS and M Popl, Analyszs of Complex Hydrocarbon Mixtures, Part B, Elsevler, Amsterdam, 1981 9 R L Miller, L S Ettre and N G Johansen, J Chromatogr ,264 (1983) 19 10 E MataovB, J KrupEik, P cellir and J Gara), J Chromatogr , 303 (1984) 151

Eva Matlsovd graduated m 1964 as Drploma Engineer m Analytreal Chemistry at the Slovak Technical Umverslty, Bratlslava In 1977she received a Ph D m Analytlcal Chemistry at the Comemus Umverslty, Bratlslava She ISnow an Associate Professor at the Department of Analytical Chemistry, Faculty of Chemical Technology, Slovak Technical Umverslty, Brattslava, Czechoslovakia Her mam interests are hydrocarbon analysis, environmental analysis and optlmlzatlon methods

Pharmaceutical analysis using fast atom bombardment mass spectrometry-mass spectrometry Steve E. Unger and Terry J. McCormick Princeton, NJ, USA

The need for the rapid elucidation of structures of pharmaceutical importance has challenged the analyst to explore new approaches Often the purity or quantity of material limits both the number and type of analyses which may be performed This is particularly true for metabohtes or isolated natural products where the sensitivity and mformation available from mass spectrometry exceeds other analytical methods In combmation with a second stage of mass analysis, mass spectrometry-mass spectrometry (MS-MS) has demonstrated its importance to the pharmaceutical analyst The idea of couplmg two or more mass spectrometers m tandem to explore the fundamental properties 0165-9936/85/$0200

of ionic dissocrations has numerous origmsiJ The mtroduction of soft ionization methods such as chemical iomzation3 greatly simphfted the mass spectrum of mixtures and together with tandem mass spectrometers4 led to the analytical exploration of MS-MS In this manner a complex mixture of components could be introduced directly mto the mass spectrometer, ionized to produce a representative species of each, and ionic parents examined by obtaming a spectrum of the mass-analyzed parent The expansion and utilization of scanmng techniques designed to elicit more spectroscopic mformation rapidly followed Simultaneous with developments m these areas were the mtroduction of desorption iomzations methods such as field desorption (FDMS), plasma desorption (PDMS), laser desorption (LDMS), secondary ion mass spectrometry (SIMS), and fast atom 0

Elsewer

Saence

Publishers B V

trendsm analyhcalchemutry, vol 4, no 7,198s

179

L

.

bombardment (FAB) It was never long before a new ionization method was coupled to a form of MSMS analysis The primary reqmrement for success was the generation of an intense ion representative of the intact molecule with preferably little or no fragmentation The first example of coupling a desorption iomzation method with MS-MS was the work of Devrenne and Diebold They obtained MS-MS spectra of organic secondary tons formed by sputtering organic hqmds and solids with a keV fast atom beam This work was followed by Bennmghoven and Sichtermann7, Day et al 8, and others using a charged primary beam, SIMS, and by Barber et al 9 who mtroduced the term fast atom bombardment and extended its application to a variety of brologically-important compounds Barber was also the first to recogmze the sigmficance of using a liquid matrix for FAB or SIMS to allow a hrgh primary ion flux to Impact the target without significant degradation of the analyte The use of MS-MS analysis of secondary ions complements and extends the potential of FAB iomzation Chemical noise common to FAB is effectively eliminated Detection limits improve considerably and enhanced fragmentation is afforded by colhsional activatron of the mass-analyzed secondary ion The reproducibihty of FAB-MS-MS spectra often allows the differentratron of isomers or the distmctron of structural features not evident from the mass spectrum alone Mixtures may be analyzed provided similar analyte sensrtivities to FAB ionization are produced There are differences between FAB or SIMS and other lomzation methods which relate to their potential for combmation with MS-MS techniques Of primary significance is the surface-sensitrve nature of the escaping secondary ion While a liquid matrix 1s generally necessary to afford a sufficiently intense secondary ion for MS-MS analysts, it presence leads to surface matrix effects which may obscure analytes m a mixture Matrix effects have been noted with several direct mixture analysrs problems resolved by MS-MS methods, however they may be most severe when usmg FAB or SIMS as the ionization method Isolates from natural products Mass spectrometry is one of the first spectroscopic methods applied to the charactertzation of natural products A survey of the isolate using a variety of ionization methods mcludmg chemical iomzatron, fast atom bombardment, and field desorption is undertaken For volatile natural products chemical ionrzatron may be preferred to FAB due to the reduction of matrix effects. For non-volatile natural prod-

ucts FAB is more amenable to MS-MS than field desorption as FDMS often fails to provtde a sufficrently intense parent ton for characterrzation Confirmation of the correct assignment of the molecular weight(s) is often provided by rdentlfymg the parent molecule under these differing romzatron conditions or from related parents in its positive and negative ion spectra The recording of both positive and negative FAB mass spectra is straight-forward as a single solvent IS capable of both protonation and deprotonation The analyst has control of the solvent (glycerol, throglycerol, dithiothreitol-dithioerythrrtol, tetramethylene sulfone, and triethanolamme are some of the more common) as well as the pH and romc strength The mtroduction of specific cations or anions to affect iomzatron often serves to identify the molecular ion, establish a fragmentation pattern, or confirm a structural assignment The mtroductron of acid or base helps enhance solubihty and affect protonation or deprotonation As isolated natural products are often peptidecontaining but seldom simple peptrdes, the ability to characterize modrfred peptrdes at low levels is essential Natural peptrdes intractable to conventronal sequencing methods have been characterized by FABMS-MS0 These are cychc or modified peptrdes not easily degraded or whose ammo acids are not evident from traditional sequencing methods. Fig 1 shows the FAB-MS-MS spectrum of the protonated molecule of His-Leu-Leu-Val-Phe-OMe and scheme 1 defines its fragmentation. The spectral quahty is good as the acqmsition time is less than 5 mm Defimtton of ummolecular dissocations not evident from the posrtive FAB mass spectrum alone are observed This improvement is due to the elimation of chemical

HIS-LEU-LEV-“AL

-PHE-0th

200

160

I20

320

360

280

240

400

440 642+ II

1

Fig I FAB-MS-MS spectrum of theprotonated molecule of HuLeu-Leu-Val-Phe-OMe

180

trends m analytical chemutry, vol 4, no 7,1985

435+e+-ew

223+--i IlOf

I ,

e-j

209+ I

336+t--l

I

I20

160

140

LEU-LEU-LEU-VAL-PHE

OMe

CATHEPSIN FAB

245+

DIGEST

MS/MS

I 463+

+

lElO+ 180

Scheme

200

220

240

tll,*

1

noise common to FAB and the enhanced fragmentation available by colhslonal activation A second feature of FAB-MS-MS m the analysis of peptldes IS the defmltlon of parentddaughter relationships which are crucial to proper sequencing and are evldent even m the presence of complex mixtures of lsolated peptldes or impurities The methyl ester HISLeu-Leu-Val-Phe-OMe 1s lsomerlc with His-LeuLeu-Leu-Phe, however, it IS easily dlstmgmshed by the loss of methanol, 610+, as well as the 180+ fragment which defined Phe-OMe from the modified Cterminus When msufflclent fragmentation 1s available from the FAB mass or MS-MS spectrum, chemical or enzymatic degratlon with subsequent direct analysis may aid m elucidating the structure or sequence For instance, Edman degradation of Asp-Tyr-(SO,)Leu-Gly-Trp-Leu-Asp-Phe-NH2 yielded 912+ and 910- parents mdlcatmg loss of the N-terminus Asp as well as the SO, group Unlike chemical degradation, enzymatic hydrolysis 1s often less vigorous No loss of SO3 from a tyrosme sulfate residue IS seen upon Cathepsm C digestion of the trlpeptlde AspTyr(SO,)-Leu The expected dlpeptlde AspTyr(S0,) as well as residual Leu were observed m the FAB mass spectrum As shown above the dlpeptldase Cathepsm C may be used to degrade a larger peptlde into its representative dlpeptldes which m turn may be examined by FAB-MS-MS After digestion of the pentapeptide Leu-Leu-Leu-Val-Phe-OMe, the protonated molecule of the intact pentapeptlde as well as its fragments dlmmlshed Additionally, there are new prominent Ions arising from the dlpeptldes Four predominant hydrolysates were seen The 245f and 243- parents arise from Leu-Leu while the 231f and 229- ions originate from Leu-Val The remaining Phe-OMe IS only observed m the positive FAB mass spectrum, (M+H)+ at 180+, due to the reduced aad-

LEU-LEU

LEU-

ILE

ILE

-LEU

ILE-ILE

Fig 2 MS-MS spectrum (top) of the 245+ ton formed after Cathepsrn C dlgestzon of Leu-Leu-Leu-Val-Phe-OMe Thrs species 1s the protonated molecule of the dcpeptlde Leu-Leu as shown by comparrson of the 180-190 a m u region with the four possible Isomers of Leu and Ile (bottom)

lty of the methyl ester There are also mdlcatlons of the partially hydrolyzed peptlde The 392+ ion IS the protonated molecule of Leu-Val-Phe-OMe formed after mltlal enzymatic cleavage Fig 2 (top) shows the spectral quality obtained upon MS-MS examlnation of one of these parents (245+) of the digest which compared well with that of the authentic dlpeptlde Small structural differences are evident from such spectra and often allow an lsomerlc asslgnment to be made The bottom of Fig 2 shows the 180-190 a m u region of the MS-MS spectra of the four possible isomers of Leu and Ile The ratio of the fragments A/B dlstmghmshes these isomers and defines the above dlpeptlde as Leu-Leu Impurities in pharmaceutical production Monobactams are p-lactam antlblotlcs which possess an N-SO, group and are active against p-lactamase producing bacteria The monobactam aztreonam IS a synthetic antlblotlc whose process development was followed using FAB-MS-MS Posltlve (top) and negative (bottom) FAB mass spectra of mother liquors obtained during development stages are shown m Fig 3 While the mother liquors are not spatially or temporally resolved as m thin-layer or liquid chromatography, the resolving power and

181

trends ln,analytrcal chemutry, vol 4, no 7,1985

POSITIVE

FAB

AZTREONAM MOTHER

LIQUOR

200

4x+

240

220

260

300

280

2 -AZTREONAM MSlMS

464

NEGATIVE

434‘

+

FAB 80

I

100

120

140

160

160

462. 440

rnh

Fig 4 Dlfferentcatron of Z- (top) from E- (bottom) Bomers of aztreonam based upon therr FAB-MS-MS spectra Ftg 3 Posrtwe (top) and negatrve (bottom) FAB mass spectra of aztreonam mother lrquors

R=H CH3 C2H5

313; 323 341

-1 I

126+I

NH NI \

I

0

RO 0

I I I I

252+

Scheme 2

trends rn analyhcal

182

I

-205

_-

‘o

4

t___--CH3

4

oJ+"< CO2H

I

I

p03-

;

,

I

I

Y, 122

CH3

311-

354~* Scheme

*--I--*

-

I -

122-

-’

3

speed afforded by mass analysts greatly exceeds that of most forms of chromatography Aztreonam yrelds a protonated and deprotonated molecule at 436f and 434-, respectrvely The presence of hrgher-molecular-weight methylene homologs of aztreonam IS mdrcated by the 450+/448- and 464+/462- parents These were established as the protonated and deprotonated molecule of the methyl and ethyl esters of aztreonam from fragmentatron evident m then MS-MS spectra (see scheme 2) A similar procedure has established the structure of numerous other rmpurities or degradants apparent m these mass spectra The presence of isomers represents a challenge to any mass spectrometrrc method and may hmrt the extent of mformatton dnectly available by MS-MS methods E- and Z-isomers of aztreonam are drstmquashed from MS-MS spectra (Fig 4) of the deprotonated molecule (see scheme 3) Most apparent are differences m the relative ran intensity of the 354fragment (SO3 loss) to the 348- fragment (C,H,02 loss) As mixtures, their spectral contrrbuttons may be resolved as linear combmatrons of then concentrations based upon these two fragments The resultmg deconvolutton using factor analysis has been accomplished with an accuracy of f5% Srmtlarly, mixtures of eptmers and dlastereomers drstmgutshed from their FAB-MS-MS spectra have been rapidly quantified by momtormg key fragments Characterization

chematry,

vol 4, ny 7,198s

Srmtlarly, glucuromdes isolated as metabohtes show the diagnostic loss of glucuromc acid (176 a m u ) FAB-MS-MS spectra of numerous glucuromde conjugates such as calcmm channel blockers and angrotensron converting enzyme mhtbrtors yield an intense metastable loss of glucuromc acid Polyene anttbrotrcs isolated from fermentation sources often contam sugars which are apparent from their loss m MS-MS spectra Amphotertcms A and B, nystatms A,, A, and A,, and polyfungin B contam the ammosugar mycosamme as evident from the intense loss of mycosamme m these spectra Polyenes which contam more than one sugar such as nystatm A, and polyfungm B also show the drstmcttve loss of the second sugar drgrtoxose The presence or absence of diagnostic neutral losses or fragments 1s a valuable mdlcator of the functtonahty or class of many pharmaceuticals These losses or fragments may be used as mdrcators of anttbrotrc or metabohte type A shift m fragment ion mass of an isolated metabohte often suggests the structural varratton to the administered drug For mstance, the angrotenston converting enzyme mhtbrtor (compound 1) was administered to subjects and then urine collected. The metabohtes were extracted mto methylene chloride after adjustment of the pH to 1 5 using aqueous hydrochlorrc acid A portion was concentrated and applied directly to the FAB matrix for analysis

\

CH3\

,CH /CH

/o I(\

CH3 0

CH3’

of functionality

Many pharmaceuticals exhlblt intense and specrftc fragmentation common to their functronahty For mstance, monobactams exhibit the loss of 80 a m u (SO, loss) m then posrtrve MS-MS spectra Desulfonation IS a useful mdrcator of the presence of a monobactam and allows the tdentlflcatron of aztreonam at low nanogram levels

The resulting negative FAB mass spectrum IS shown m Fig 5 (top) along with the proposed structures for the two major metabohtes for 1 The MS-MS spectrum of the 434- parent (shown below) compares well with the authentic acid Its salt was also evident at m/z 456 The apparent oxygenatton

183

trendsWIanalytrcalchemrstry,vol 4, no 7, I985

NEGATIVE -

ACID

onstrated rts utthty to produce ions of non-volattle, thermally-labile compounds its coupling wrth MS-MS methods and rts apphcatrons to mixture analysis will continue to yield success

FAB

EXTRACT

URINE

50

250

I50

References R G Cooks, J H Beynon, R M Capnoh and G R Lester, Metastable Zons, Elsevler, New York, 1973 J H Futrell and T 0 Tlernan, m J L Frankhn (Editor), Ion Molecule Reactions, Plenum, New York, 1972, Chap 11 M’ S B Numson and F H Field, J Am Chem Sot , 88 (1966) 4337

FAB

’

MS/MS

450-e

F W McLafferty (Editor), Tandem Mass Spectrometry, John Wiley, New York, 1983 K L Busch and R G Cooks, Science, 218 (1982) 247 F M Devlenne and A Dlebold, C R Hebd Seances Acad Scr ,278 (1974) 1219 A Benmnghoven and W Slchtermann, Anal Chem , 50

253-

434-

(1978) 118

R J Day, S E Unger and R G Cooks, Anal Chem , 52 (1980) 557A

__--------_ M-?_”

200

220

240

9 M Barber, R S Bordoh, R D Sedgwlck and A N Tyler,

260

Nature, 293 (1981) 270

10 K B Tomer, F W Crow, M L Gross and K D Kopple, Anal Chem ,56 (1984) 880

5 360

Steve E Unger and Terry J McCormick are at the Squibb Znshtute for Medical Research, P 0 Box 4000, Princeton, NJ 08540, USA

380

400

I?!/*

Fig 5 Negative FAB mass spectrum (top) of a unne extract shows two metabohtes at m/z 434 and 450 The FAB-MS-MS spectrum (bottom) &&rates a 16 a m u shift m the malor fragment and suggests the srte of fragmentation

In Forthcoming Issues A program for robust cahbratlon usmg a combmatory algonthm for regression by Max Femberg

Trends m computer user-interface

technology

by T C O’Haver

product was observed at m/z 450 The MS-MS spectrum of the 4150~parent showed a 16 a m u shaft m the 237- fragment to 253- (see insert) Thus fragrnentatron occurs at the peptrde linkage and defines the site of oxygen mcorporatron as on the phosphemc acid moerty Conclusions As a better understandmg of the solution and mterfacral chemistry of pharmaceuttcal mrxtures develops, approprrate modrftcatrons or adjustments to the FAB matrix will yield a more intense or more representative ion populatton for MS-MS analysis The key to mass spectrometry IS the generatron of tons without which no degree of sophisticated mstrumentatron will afford a result Since FAB has dem-

The next stage m laboratory robotics by T Hlrschfeld

Correlation gas chromatography degradation studies

m polymer thermal

by M Kallurand, E Kulhk

Modern analytical methods m connection with organic synthesis by L Johansson

Progress m TLC by C Poole

High performance preclpltatlon by G Glockner

hqmd chromatography