Alloxazines and isoalloxazines. Mass spectrometric analysis of riboflavin and related compounds

Organic Mass Spectrometry, 1972, Vol. 6, pp. 1383 to 1399. Heyden & Son Limited. Printed in Northern Ireland

ALLOXAZINES AND ISOALLOXAZINES. MASS SPECTROMETRIC ANALYSIS OF RIBOFLAVIN

AND RELATED COMPOUNDS PETER BROWN, CECIL L. HORNBECK and JOHN R. CRONIN

Department of Chemistry, Arizona State University, Tempe, Arizona 85281, USA

(Received 4 April 1972; accepted (revised) 30 June 1972)

Abstract-The positive ion electron-impact mass spectra of a series of alloxazines, iso-alloxazines and some derivatives have been examined. The compounds employed were lumichrome (7,8- dimethylalloxazine), 1,3-dimethyllumichrome, lumiflavin (7,8,10-trimethyl-iso-alloxazine), 3- methyllumiflavin, riboflavin [7,8-dimethyl-lO-(D-l’-ribityl)-iso-alloxazine], riboflavin tetraacetate, 3-methylriboflavin tetraacetate and riboflavin tetrapropionate. By using exact mass measurements, metastable ion defocusing and the mass/composition shifts occurring with derivatives, it has been possible to arrive at detailed interpretations of the mass spectra of all compounds. With lumichrome and lumiflavin, fragmentation commences by elimination of HNCO from the pyrimidine ring. With riboflavin and its derivatives the ribityl chain cleaves off first, followed by decomposition of the iso-alloxazine ring. Application of these methods and findings to the structural analysis of chemically interesting modified flavins is predicted to be rewarding.

I N T R O D U C T I O N

RIBOFLAVIN [7,8-dimethyl-lO-(D-1’-ribityl)-iso-alloxazine] contributes the operative redox system in the coenzymes FMN (flavin mononucleotide) and FAD (flavin adenine dinucleotide). These coenzymes are incorporated alone and with other cofactors into flavoproteins, which catalyze a variety of essential oxidative reactions in viuo. In some flavoproteins’ to the coenzyme is covalently linked to the protein. In these cases enzymatic hydrolysis, acid hydrolysis and various other treatments yield flavins to which remain attached, respectively, p e p t i d e ~ , l * ~ * ~ amino acids1 and various other fragment^.^

Riboflavin itself undergoes many intriguing chemical reactions, e.g. photolysis to afford mainly lumichrome (7,s-dimethylalloxazine) under neutral or acidic conditions and primarily lumiflavin(7,S,lO-trimethyl-iso-alloxazine) under basic conditiom6

Our interest in the structure of these materials and in their chemical properties as these relate to the mechanism of action of the parent flavoenzymes has prompted exploration of convenient experimental methods for structure elucidation on a sub- milligram scale. To this end, a systematic investigation of a series of alloxazines and iso-alloxazines has been made by positive ion electron-impact mass spectrometry, utilizing both low and high resolution data, metastable ion defocusing’ and the ‘shift technique’.s Since riboflavin itself is rather involatile and decomposes at about 290°, acetate and propionate esters and N-methyl derivatives were employed both to increase volatility and to permit effective application of the ‘shift technique’.8 Apparently, no such studies have been reported previously in the literature9~l0 although an abstractll briefly describes similar studies, and isolated examples such as the negative ion spectra of riboflavin12 and 7,8-dimethyl-l0-formyl-methyl-iso-alloxazine ethyl hemiacetaP3 have appeared. In the latter paper13 the authors assert that peaks higher than the molecular weight were recorded in their attempts to obtain positive ion spectra of these compounds, and further, that the positive ion spectra which they

1383

1384 P. BROWN, C. L. HORNBECK and J. R. CRONIN

R

(I) R = H Lumichrome (11) R = CH, 1,3-Dirnethyllumichrome

(111) R = H Lumiflavin (IV) R = CH, 3-Methyllumiflavin

0 (V) R, = R, = H Riboflavin

Riboflavin tetraacetate 3-Methylribaflavin tetraacetate

(VI) R, = CH,CO, R, = H (VII) R, = CH,CO, R, = CH,

(VIn) R, = CH,CH,CO, R, = H Riboflavin tetrapropionate

did secure were too complex to interpret.13 Neither of these difficulties arose in the work that we describe here. Mass spectral studies of other nitrogen-containing heterocyclic systems such as pyrimidines,14 purines14 and pteridines,l4,l5 and also of monosaccharide derivatives such as acetate$ have clearly demonstrated that much structural information can be extracted from this approach.

DISCUSSION We report here and discuss in some detail the electron-impact spectra of the allox-

azines lumichrome (I, Fig. 1) and 1,3-dirnethyllumichrome (11, Fig. 2); of the simple iso-alloxazines lumiflavin (111, Fig. 3) and 3-methyllumiflavin (IV, Fig. 4); of the flavin riboflavin (V, Fig. 5) and of its derivatives riboflavin tetraacetate (VI, Fig. 6), 3-methylriboflavin tetraacetate (VII, Fig. 7) and riboflavin tetrapropionate (VIII, Fig. 8). The elemental compositions of selected fragment ions in the spectra of compounds I , 11, 111, IV and VI were determined by accurate mass measurements using an apparent resolution of 15,000 and are displayed in Tables 1 to 5.

The major decomposition pathways of lumichrome (I, Fig. 1, Table 1) on electron- impact are given in Scheme 1.” It is apparent that fragmentation is initiated by expulsion of the elements of HNCO from the tautomeric pyrimidine ring, followed by the elimination of CO and CH,, thus accounting for the species at m/e 199, 171 and

Alloxazines and isoalloxazines 1385

L U f l l C n R O f l E (300%)

W c

I = I 1 4

0. 156 .-

0. I71

I C I 2 H l O O 2 N 4

v) L W c

5:. 13 199 (M-43)

105 2 D I I 58 79 L

0. n I

(M-15) 1 (M-28)227 I J I I I 214

2s 50 7s 100 12s 150 l i 5 200 25s

13

'i'

FIG. 1. Mass spectrum (70 eV) of lumichrome (I)

TABLE 1. ELEMENTAL COMPOSITIONS OF FRAGMENT IONS : LUMICHROME (I)

nile Composition mle Composition

1 +*

(1) C,,H,,N,O, m/e 242 [MI ni/e 199 C,,H,N,O

b A .N. r \ . I\ ..

mle 144 m / e 171 C,,H,N, m/e 156 C9HGN3 SCHEME 1

156. With 1,3-dirnethyllumichrome (11, Fig. 2, Table 2), expulsion of CH,NCO from the molecular ion occurs first, followed by loss of CO and CH, (Scheme 2).* This produces an analogous series of peaks at m/e 213 (70%), 185 and 170 that are shifted to higher mass by 14 amu (CH,), presumably because of retention of the N-1 methyl group in these ions as depicted in Scheme 2.

* The observation of metastable ion peaks is indicated in the Schemes as in*.

7 4 2 0 PP.)


(200%) 1.3-DlMETHYLLUnlCHROnE

FIG. 2. Mass spectrum (70 eV) of 1,3-dimethyllumichrome (11).

TABLE 2. ELEMENTAL COMPOSITIONS OF FRAGMENT IONS: 1 &DIMETHYL- LUMICHROME (11)

in/e Composition mle Composition

In addition, 1,3-dimethyllumichrome (11) exhibits prominent ion peaks because of elimination of CO, CHO and a further CO, giving rise to species at m/e 242, 241 and the remainder (30'4 of 213 (Scheme 2). Plausible structures are also suggested for the m/e 158 and 131 ions and proposals made for their origin and fate.

The iso-alloxazines lumiflavin (111, Fig. 3, Table 3) and the derivative 3-methyllumiflavin (IV, Fig. 4, Table 4) behave somewhat similarly (Scheme 3). The principal ions and their compositions can be conveniently rationalized in terins of successive losses from the molecular ions of the neutral species RNCO (R = H for 111, R = CH, for IV), CO, CH, and HCN. Examination of the mass spectra of the alloxazine and iso-alloxazine structural isomers 1,3-dimethyllumichrorne (11, Fig. 2) and 3-methyllumiflavin (IV, Fig. 4) reveals few differences in terms of ion peaks and their elemental compositions although considerable intensity differences can be discerned. The former is explicable in terms of the fragmentation pathways postulated in Schemes 2 and 3, in that methyl groups at N-1 (11) and N-10 (IV) tend to be retained, whereas substituents at N-3 are lost at an early stage.

In Fig. 5 is reproduced the 70eV mass spectrum of riboflavin (V), taken at a direct probe temperature of 260°, in which the molecular ion peak (m/e 376) is clearly in evidence while significant peaks at higher mass are not. Ion peaks at m/e 315 and 285 can be explained by simple cleavages in the ribityl side-chain between


SCHEME 2

L U t l I F L f l V I N

256 (Ul

FIG. 3. Mass spectrum (70 eV) of lumiflavin (111).


TABLE 3. ELEMENTAL COMPOSITIONS OF FRAGMENT IONS : LUMIFLAVIN (111)

mle Composition mle Composition

m/e 198 80:: C,,H8N30 1 1

-co~"l*

V

m/e 185 CllHllN3

SCHEME 3

3-IlETHYLLUHlFLAVIN FH3

tl@SS/CHRRGE

FIG. 4. Mass spectrum (70 eV) of 3-methyllumiflavin (IV).


RIBOFLAYIN

1

FIG. 5. Mass spectrum (70 eV) of riboflavin (V).

C-3‘/C-4’ and C-2’/C-3’ respectively; whereas species at m/e 256 and 242 can be generated by cleavages between C-l’/C-2’ and N-lO/C-l’, respectively, with one net hydrogen transfer and the ion at m/e 243 by cleavage between N-lO/C-l’ with two hydrogens transferred. Based on elemental composition data for riboflavin tetraacetate (VI, Table 5) the ions of mass 256 and 242 in the spectrum of V also are iso- meric with the molecular ions of lumiflavin (111) and lumichrome (I), respectively. Since the peaks at m/e 242,243 and 256 are the most intense in the spectrum of riboflavin (V, Fig. 5), the fact that virtually every ion peak below m/e 242 can also be found in the spectrum of lumichrome (I, Fig. 1) and/or lumiflavin (111, Fig. 3) comes as no surprise. In summary, the ribityl moiety is readily lost from the molecular ion with the charge residing preferentially on the heterocyclic ring system.

The mass spectrum of the derivative riboflavin tetraacetate (VI, Fig. 6), taken at the lower probe temperature of 165”, is more complex and contains much information concerning the fragmentation of the acetylated monosaccharide entity. Inter- pretation of this spectrum was achieved by using extensive accurate mass measurements (Table 5) and also peak shifts observed in the spectra of 3-methylriboflavin tetraacetate (VII, Fig. 7) and riboflavin tetrapropionate (VIII, Fig. S), as displayed in Table 6. Metastable defocusing experiments provided evidence for consecutive stages of stepwise decomposition pathways (m* in Schemes).


TABLE 5. ELEMENTAL COMPOSITIONS OF FRAGMENT IONS : RIBOFLAVIN TETRAACETATE (VI)

rnle Composition mle Composition

FIG. 6. Mass spectrum (70 eV) of riboflavin tetraacetate (VI).

As an example, the molecular ion of riboflavin tetraacetate (VI, Fig. 6) occurs at m/e 544 but is shifted by 56 (4 x 14) amu to m/e 600 (Table 6) with the tetrapropionate (VIII, Fig. 8). As indicated in Scheme 4, the molecular ion of the tetraacetate (VI) produces ions at m/e 502, 501, 486 and 485 by direct losses of the neutrals RCHCO, RCH,CO, RCHCOz and RCH2COZ* (R = H), respectively.? The corresponding ions appear at m/e 544, 543, 528 and 527 with the tetrapropionate (VIII) because of loss of analogous neutrals (R = CH,), each of these peaks shifted now by 42 (3 x 14) amu (Table 6). Thus the ions at 7n/e 502,501,486 and 485 in the spectrum of riboflavin tetraacetate (VI) must contain only three acetoxy groups," entirely consonant with the elemental composition data (Table 5 ) , and fragmentation must be proceeding in the acylated ribityl side chain rather than in the iso-alloxazine ring system. This picture

* The data presented here does not indicate specifically which acyl group(s) is (are) fragmenting first, but only the number remaining intact and the number of carbon atoms left in the ribityl entity. The structures in the Schemes are intended to depict the level of fragmentation only.

t Absence of an [M - 601 peak (because of loss of acetic acid) is at first sight surprising. Such behavior has been noted beforei5 with monosaccharide peracetates, however, where the principal peak in this area is at [M - 591.


TABLE 6. MASS SHIFTS OF ANALOGOUS IONS IN THE MASS SPECTRA OF RIBOFLAVIN TETRAACETATE (VI), 3-METHYLRIBOFLAVIN TETRAACETATE (VII) AND RIBOFLAVIN TETRAPROPIONATE (VIII)

(VW (VI) (VIII) (Vw wu (Vm

256 242 242 381 367 381 257 243 243 383 3 69 371 (?)

389 375 375 270 256 256

282 268 268 40 1 387 40 1 295 281 281 413 399 413 299 285 285 41 5 40 1 415 311 297 297 43 1 417 431

439 425 453 - - 299

281 267 267 397 383 397

322 308 308 440 426 454 325 311 311 443 429 457 329 315 315 456 442 470 337 323 323 457 443 471 341 327 327 473 45 9 487

499 485 527 353 339 339

500 486 528 355 341 341 359 345

373 359 359 558 [MI 544 [MI 600 [MI

345 515 501 543 371 357 357 516 502 544

is fully confirmed with 3-methylriboflavin tetraacetate (VII, Fig. 7) where the equiv- alent ions all move up by 14 amu to m/e 516,515,500 and 499 (Table 6). Application of this type of reasoning allows the interpretation of almost every peak in the spectrum of VI.

Plausible structural representations are given in Scheme 5 for species at m/e 459, 443, 442, 429, 426 and 425 present in the mass spectrum of the tetraacetate (VI), and their precursor ions. These peaks are shifted by 28 (2 x 14) amu to m/e 487, 471, 470, 457, 454 and 453 (Table 6) respectively with the tetrapropionate (VIII), clearly corroborating the high resolution data (Table 5) for the tetraacetate (VI), which indicates that only two acetoxy moieties remain in this group of ions.

In Scheme 6 are displayed ions observed in the spectrum of tetraacetate (VI) at m/e 417, 401, 399, 387 and 383, and their possible precursors. This series of ions is shifted uniformly by 14 amu to m/e 431,415, 413, 401 and 397 (Table 6) with the tetrapropionate (VIII), and therefore must possess but a single acyloxy group.

Reasonable structural representations for ions in the spectrum of tetraacetate (VI) at mle 375, 357, 341, 339 and 323 are given in Scheme 7, and at m/e 345, 327, 315, 297, 285 and 281 in Scheme 8. None of these peaks is shifted at all* (Table 6) in the spectrum of the tetrapropionate (VIII), implying that all acyloxy groups have now been detached at this level of fragmentation.

Important peaks occur in the mass spectrum of riboflavin tetraacetate (VI) and also riboflavin (V) itself at m/e 256, 243 and 242, that are unshifted in the tetrapropionate (VIII) spectrum but move up to m/e 270,257 and 256 with 3-methylriboflavin tetraacetate (VII, Fig. 7, Table 6) . This information, coupled with elemental composition data (Table 5) for the tetraacetate (VI), suggests structures for the m/e 242 and

* Actually, a portion of m/e 357 seems to move up to 371 with the tetrapropionate (VIII) and all m/e 367 shifts to 381 (Table 6).



Ac OAc & Ii

mNcxo N'

w m:,qxo 0

nile 426 C,,H,,N,O,

-AcOH m*

rule 486 \

SCHEME 5

II 0

ni/e 425 C,,H,,N,06

-*coF\,\*o

ni/e 485 443

255 species closely related to the molecular ions of lumichrome (I) and lumiflavin (II), respectively, as envisaged in Scheme 9. From the general appearance of the spectrum and from exact mass measurements (Table 5) peaks below m/e 242 discerned with riboflavin tetraacetate (VI) are apparently generated by further decomposition of the nzle 256, 243 and 242 species (See Schemes 1 and 3).

From the observed peak shifts (Table 6) and elemental compositions of fragment ions (Table 5) present in the spectrum of riboflavin tetraacetate (VI), it is obvious that

7A

1394 P. BROWN, C. L. HORNBECK and J . R. CRONIN

-CHzCO III* 1 -CH,CO 1 -

mle387 C,, m/e 383 ClsH,!,N40,

-CH,CO m* 1 mfe 429 mnle485 425

SCHEME 6

decomposition of the acylated ribityl side-chain precedes that of the iso-alloxazine entity.* For example, m/e 501 [M - 431 is not caused by loss of HNCO [as with lumiflavin (111) (Scheme 3)] but is due to expulsion of CH,CO, presumably from an acetoxy group. In fact every ion examined including and above m/e 242 in the spectrum of VI still contains the four nitrogen atoms of the iso-alloxazine ring system (Table 5).

The actual structures of any ions discussed in this work of course remain completely unknown. The 'structural representations' set out in the Schemes are meant solely to illustrate the level of fragmentation in a more graphic way than simply reproducing only the molecular formulae of the ions. With the acyl derivatives, there is no evidence as to which acyl group(s) fragmentation(s) is (are) occurring in at any particular step except where limited by the number of carbon atoms remaining of the ribityl chain. Also, in the absence of isotope labeling experiments detailed mechanisms are not to be inferred from the schemes.

Examination of Schemes 4 to 8 shows that variations in the structures of the N-10 polyol substituent should be accessible from ion peak and composition shifts secured with a suitably altered flavin. Thus mass spectrometry might be expected to be useful

* Preferential fragmentation of the saccharide part of the molecule also happens with l-phenyl- flavazole derivatives" and with cardiac glycosides.l* Where polysaccharides are involved, the spectrum contains information betraying the number and sequence of monosaccharide units present.

OH OH

0 0 m/r 375 C,,HlgN,O,j m/e 357 C,,H,,N~O~

-AcO{ -cn,co \k m/e 417 399 375 m[e 417

l??/C 375

II 0

mle 327 c16

-H,O 1 mle 345

m/e 339 C17

-..Of '\-H~o

SCHEME 7

mle399 357

0 mle 315 CIS

mle 345

0

6 m/e 341 C,TH,iN,O,

-AcOA 1 mle 401

m/e 323 C,,H,,N,O,

-AcOH 1 m/e 383

0 mle 297 CIS

-H20 1 m/e 315

SCHEME 8 1395


:I i

c - -

FIG. 8. Mass Spectrum (70 cV) of Riboflavin Tetrapropionate (VIII).

OAc OAc W A C

p H > H CH2 m:qf - jT&yy N U

: : : F o * c

2 LH ,yJf,y$+mtz-";." N' m;,y-x0 -

0 0 (V) I H / E 256 CiSI-I1$,0,

0 0 nr/e 242 m/e 243

C,,H,,N,OZ ClJ J,LN,O, 0

(V)

SCHEME 9

in further characterization of intermediates in the photolysis6 of riboflavin to lumiflavin and lumichrome, and in structural elucidation of unknown flavins suspected to differ from riboflavin in the N-10 side chain.lg

Furthermore, since the side chain is readily lost from the molecular ion, a series of peaks derived from breakdown of the heterocyclic ring system is seen in all cases. The composition and intensity of these peaks should be characteristically affected by substitution of the ring and be diagnostic of both the nature and position of the substituent. This raises the further possibility of structural characterization of synthetic flavin analogs, naturally occurring substituted flavinsl to and intermediates in enzymatic reactions and enzymatic model studies20s21 when sufficiently stable.

Finally, it is of great practical importance to emphasize the sensitivity of the approach described here and the enormous amount of structural information available from a small quantity of sample. For example, 100 nmoles of riboflavin (V) have been successfully acetylated to VI," which was quite sufficient to produce an intense and characteristic mass spectrum.

* R. Hendriks, unpublished observations.


E X P E R I M E N T A L Mass spectra

All spectral data were obtained by Mr Richard Scott. Low resolution spectra were recorded on a Varian MAT SMlB double focusing instrument operating under the following conditions: trap current 300 pA, electron energy 70 eV, accelerating voltage 8 kV, resolution approx. 1000, source temperature 175", direct inlet water-cooled probe temperature as listed in Table 7. Accurate mass measurements were made under similar conditions by using the peak matching technique with pfk as standard except that resolution was set at approx. 15,000.* After decade box calibration measurements were made at least in duplicate. The ratios were averaged and entered together with the reference masses into computer program ELCOMP, written by Mr Edwin Black, the output of which is: (i) all possible C,H,O,N, combinations and their masses within a given (adjustable) mass limit, e.g. k0.005 amu in this instance, (ii) the observed mass of the unknown peak and (iii) the mass errors between calculated and observed masses. Inspection of the tabular print-out and disqualification of chemically impossible combinations invariably showed only one composition in contention for the least mass error. Doublets were treated as two separate peaks in this procedure.

Metastable ion defocusing' was achieved in the first drift region between source and electrostatic sector, by using a continuously variable (3 to 12 kV) accelerating voltage control unit.2z

Low resolution mass spectra were plotted by a CalComp plotter, using program BGRAPH written by Dr William D. Lucky.

TABLE 7. DIRECT INLET PROBE TEMPERATURES

Compound Temp.

Lumichrome (I) 1,3-Dimethyllumichrome (11) Lumiflavin (111) 3-Mcthyllumiflavin (IV) Riboflavin (V) Riboflavin tetraacetate (VI) 3-Methylriboflavin tetraacetate (VII) Riboflavin tetrapropionate (VIII)

140 115 175 160 260 165 150 160

Compounds Melting points were obtained with a Thomas Hoover capillary melting point apparatus and

were corrected from a calibration curve prepared with commercial melting point standards. Products were dried under vacuum at 130" prior to melting point determinations. Descending paper chromatography was carried out on Whatman 1 paper by using the lower phase of n-butano1:acetic acid: water (40: 10:50 v/v) as developer. Ascending thin layer chromatography was carried out on air dried 300 micron silica gel (Camag D5) plates by using n-butano1:acetic acid:water (80:20:20 v/v) and benzene:methanol:acetone:acetic acid (70:20: 5 : 5 v/v) as developers. Flavins were spotted in 1 to 3 pg amounts and detected by their fluorescence in ultraviolet light. All reagents were reagent grade or better and used without further purification.

Riboflavin was purchased from Sigma Chemical Co and recrystallized from water (decomposes 289 to 291", Litz3 290"). Lumichrome was purchased from Aldrich Chemical Co and recrystallized from pyridine (melting point above 365", Litz4 > 300"). Lumiflavin was synthesized according to the method of Hemmerich et aLZ5 The product decomposed around 325" (Lit.25 322").

Riboflavin tetraacetate and riboflavin tetrapropionate were synthesized from riboflavin by the method of McCormick,Z6 substituting propionic acid/propionic anhydride for acetic acid/acetic anhydride in the latter case. The product was recrystallized repeatedly from a minimum volume of dichloromethane and five volumes of petroleum ether until a homogeneous yellow-orange powder was obtained. The tetraacetate melted with decomposition at 238 to 242" (Lit.27a'2ib 242 to 246") and the tetrapropionate melted from 188 to 189" (Lit.Zia 186 to 187").

* Accuracy approx. 5 ppm.


1,3-Dimethyllurnichrome, 3-methyllumiflavin and 3-methylriboflavin tetraacetate were synthesized, respectively, from lumichrome, lumiflavin and riboflavin tetraacetate by an adaptation of the per- methylation procedure of Das et aLZR A saturated solution of the starting material in 100 ml N,N- dimethylformamide (DMF) was prepared. With lumiflavin and riboflavin tetraacetate 150 mg sodium dichromate were added to the DMF prior to dissolution of the flavin. Silver oxide (2 gm) and methyl iodide (10 ml) were added and the reactions allowed to proceed overnight with shaking at room temperature. The reactions were terminated by adding 40 ml DMF, filtering and extraction of the products into 400 ml chloroform. The chloroform was washed with 300 ml 5 % potassium cyanide solution. After discarding the aqueous layer, the organic phase was washed exhaustively with water, dried twice with anhydrous sodium sulfate, filtered and the chloroform evaporated under a stream of dry nitrogen gas at approximately 50". The residue was repeatedly recrystallized from dichloromethane and petroleum ether and dried under a water aspirator. 1,3-Dimethyllurni- chrome was a light yellow powder melting at 251 to 252". 3-Methyllumiflavin and 3-methylriboflavin tetraacetate were both yellow-orange powders, the former decomposing around 290" and the latter decomposing around 175" (Lit.a7b3z7c 185 to 191").

All the materials described above were homogeneous when analyzed by paper and thin layer chromatography.

Acknowledgements-We are indebted to the National Science Foundation for financial assistance (Grant No. GP-6979) in acquiring the Varian MAT SMlB mass spectrometer and to the US Public Health Service for a grant in support of this work (AM 12908).

R E F E R E N C E S 1. W. H. Walker and T. P. Singer, J. Biol. Chem. 245,4224 (1970). 2. W. H. Walker, E. B. Kearney, R. Seng and T. P. Singer, Biochem. Biophys. Res. Commun. 44,

287 (1971). 3. D. R. Patek and W. R. Frisell, Arch. Biochem. Biophys. submitted for publication. 4. R. G. Bartsch, T. E. Meyer and A. B. Robinson, in K. Okunuki, M. D. Kamen and I. Sekuzu

(Eds.), Structures and Function of Cytochromes, University Park Press, Baltimore, Md, 1968, p. 443.

5. R. Hendriks and J. R. Cronin, Biochem. Biophys. Res. Commun. 44,313 (1971). 6. W. L. Cairns and D. E. Metzler, J. Am. Chem. Soc. 93,2772 (1971). 7. M. Barber and R. M. Elliott, ASTM Committee E-14, Abstracts of 12th Annual Conference on

Mass Spectrometry and Allied Topics, Montreal, June 1964, p. 150. 8. K. Biemann, Mass Spectrometry: Organic Chemical Applications, McGraw-Hill Inc, New

York, 1962, p. 309. 9. G. E. Van Lear and F. W. McLafferty, Ann. Rev. Biochem. 38,289 (1969).

10. C. Fenselau, in C. F. Chignell, (Ed.), Methods in Pharmacology, Vol. 2, Physical Methods,

11. 0. Gawron and K. P. Mahajan, Federation Proc. 28,853 (1969). 12. M. von Ardenne, K. Steinfelder and R. Tummler, Z. Chem. 5,287 (1965). 13. R. Tummler, K. Steinfelder, E. C. Owen and D. W. West, Org. Mass Spectrom. 5,41 (1971). 14. H. Budzikiewicz, C. Djerassi and D. H. Williams, Mass Spectrometry of Organic Compounds,

15. (a) J. A. Blair and C. D. Foxall, Org. Mass Spectrom. 2,923 (1969); (b) P. Haug, Org. Mass

16. (a) K. Biemann, D. C. DeJongh and H. K. Schnoes, J. Am. Chem. SOC. 85, 1763 (1963); (b)

17. G. S. Johnson, W. S. Ruliffson and R. G. Cooks, Chem. Commun. 587 (1970). 18. P. Brown, F. Bruschweiler, G. R. Pettit and T. Reichstein, Org. Mass Spectrom. 5 , 573 (1971). 19. K. Matsui, in D. B. McCormick and L. D. Wright (Eds.), Methods in Enzymology, Vol. XVIII,

20. G. A. Hamilton and L. E. Brown, J. Am. Chem. SOC. 92,7225 (1970). 21. W.-R. Knappe and P. Hemmerich, Federation ofEuropean Biochemical Societies Letters 13,293

22. P. Brown, Org. Mass Spectrom. 3, 1175 (1970).

Appleton-Century-Crofts Inc, New York, 1972.

Holden-Day Inc, San Francisco, Ca, 1967, Chapter 21.

Spectrom. 3, 1365 (1970).

K. Heyns and H. Scharmann, Ann. 667, 183 (1963).

Academic Press, New York, 1971, p. 433.

(1971).


23. (a) J. A. Means, Th. C. Grenfell and F. H. Hedger, J. Am. Pharm. Assoc. 32,51 (1943); (b)

24. R. M. Cresswell and H. C. S. Wood, J. Chern. SOC. 4768 (1960). 25. P. Hemmerich, S. Fallab and H. Erlenmeyer, Helu. Chim. Acta 39, 1242 (1956). 26. D. B. McCormick, J. Heterocyclic Chem. 7, 447 (1970). 27. (a) Tokyo Tanabe Pharmaceutical Co Ltd, Chem. Abstr. 71, 124504~ (1969); (b) Y. Kyogoku

and B. S. Yu, Bull. Chem. SOC. Japan 42, 1387 (1969); (c) P. Hemmerich, Helv. Chim. Acta 47, 464 (1964).

S. Shimizu, Chem. Abstr. 51,15638a (1957).

28. B. C. Das, S. D. Gkro and E. Lederer, Biochem. Biophys. Res. Commun. 29,211 (1967).

Documents

Alloxazines and isoalloxazines. Mass spectrometric analysis of riboflavin and related compounds