mass spectrometry

ANALYTICAL

BIOCHEMISTRY

181,302-308

(1989)

The Identification of 5,6-Dihydrouridine in Normal Human Urine by Combined Gas Chromatography/ Mass Spectrometry’ Mark L. J. Reimer,* Karl H. Schram,*,2 Katsuyuki Nakano,? and Toshio Yasaka? *Department of Pharmaceutical Sciences, College of Pharmacy, University of Arizona, Tucson, Arizona TPL Comprehensive Research Institute, 1 Kamiyamacho, Tondabayashi, Osaka, 584, Japan

Received

April

6,1989

The identification of 5,6-dihydrouridine in normal human urine is reported. Partial purification and isolation of the compound by boronate gel affinity chromatography and reversed-phase high-performance liquid chromatography preceded its characterization as a trimethylsilyl derivative by combined gas chromatography/mass spectrometry. Structure proof is based upon a comparison of mass spectral and chromatographic features of the urinary component to that of an authentic reference sample. Additional data derived from high resolution mass measurements and deuterium isotopelabeling experiments provide confirmation of fragment ion structure. The poor detectability inherent in the HPLC/uv analysis of nucleosides is also discussed. 0 1X39 Academic

Press, Inc.

5,6-Dihydrouridine (D)3 (Fig. 1) was first reported as a naturally occurring component of yeast tRNAAla by Madison and Holley in 1965 (l), although subsequent studies have shown it to be present in the tRNAs of most organisms (2). As with other modified nucleosides, D is biosynthesized post-transcriptionally, in this case by hydrogenation of the C5-C6 double bonds of selected uridines. Second in abundance to pseudouridine among the modified nucleosides of tRNA, 5,6-dihydrouridine is lo’ Supported by Grant CA43068 from the National Institutes of Health (K.H.S.) and by a grant from Patriarch Takahito Miki and the Church of Perfect Liberty, Japan (K.N.). * To whom correspondence should be addressed. ’ Abbreviations used D, 5,6-dihydrouridine; SV, simian virus; tsA, ~-[9-(~-D-ribofuranosyl)purin-6-yl-carbamoyl]-L-threonine; U, uridine; Tm, 2’-0-methylribosylthymine; TMS, trimethylsilyl, BSTFA, N,O-bis(trimethylsilyl)trifluoroacetamide; TMCS, trimethylchlorosilane; PFK, perfluorokerosine; PNU, pooled normal urine; miA, lmethyladenosine; RIC, reconstructed ion chromatogram, RI, relative intensity; S, sugar; B, base; EI, electron ionization. 302

85721, and

cated primarily in the left-hand or “dihydrouridine loop” (especially positions 16, 17, and 20), with an additional residue occasionally appearing in loop 3 (often after m7G) (3). Despite its ubiquitous nature, the biological function of D remains unclear (4). Cerutti and Miller have proposed that a reversible conversion of uridine residues into D could represent a means for regulating the activity of the particular tRNA molecule (5). X-ray crystal studies (6-8) suggest that the saturated ring structure of 5,6-dihydrouridine may modify neighbor-neighbor base stacking interactions, which could in turn influence the tertiary structure of the D loop. Reductions of 5,6-dihydrouridine levels in mice have been indirectly linked to cancer diagnosis and treatment. In one of the few structural studies to document virally induced changes in tRNA modification, Raba and co-workers (9) sequenced a tRNALys appearing in SV40transformed mouse fibroblasts and found it to be hypomodified compared to the corresponding rabbit liver tRNALys. In addition to finding A37 instead of t6A37 and U, T, or & instead of Trnsd, sequence analysis revealed a IJZOresidue instead of the customary DZ,,. In another study, mouse tRNA isolated from tumorous, 5-fluorouracil-treated mammary tissue showed a reduction in the amounts of D, T, and IJ compared to normal tissue (lo), suggesting that the administration of this widely used cancer chemotherapy agent may inhibit the enzymes catalyzing the biosynthesis of these nucleosides. Interestingly, one of the most important metabolites of 5-fluorouracil is 5,6-dihydro-5-fluorouracil; GC and GC/ MS techniques for the quantitative analysis of these compounds in plasma have been described (11,12). The absence of specific enzyme systems in the eucaryotic cell prohibits the incorporation of modified nucleosides into macromolecular nucleic acids. Consequently, in the natural course of mammalian tRNA catabolism, 0003-2697/89

$3.00

Copyright 0 1989 by Academic Press, Inc. All rights of reproduction in any form reserved.

DIHYDROURIDINE

BY

GAS

CHROMATOGRAPHY/MASS

SPECTROMETRY

303

Pure Chemical Industries, Ltd. (Osaka, Japan). Pyridine (silylation grade) and N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) containing 1% trimethylchlorosilane (TMCS) were obtained from Pierce Chemical Co. (Rockford, IL). BSTFA-2H1s (DeuteroRegisil-d”) was obtained from Regis Chemical Co. (Morton Grove, IL). An authentic sample of 5,6-dihydrouridine was purchased from Sigma Chemical Co. (St. Louis, MO). A Milli-Q Reagent Water System (Millipore, Bedford, MA) was used for water purification.

HO FIG. 1.

OH 5,6-Dihydrouridine.

many of the modified nucleosides are either metabolized or excreted intact in urine (13,14). Extensive research has established that the levels of certain modified nucleosides excreted in the urine of cancer patients are markedly elevated compared to values found in normal human urine (15-26). Such a process has enormous potential as a noninvasive biological marker for the diagnosis, treatment, and long-term monitoring of various forms of neoplasia. While the majority of these studies have relied solely upon HPLC/uv techniques for nucleoside separation and identification, considerable attention has recently been given to mass spectral-based methods (14,27-29), including those involving capillary GC/MS (30). Before significant progress can be made in characterizing novel nucleosides in cancer patient urine, baseline levels in normal urine must be established. Toward this end, we report the identification of 5,6-dihydrouridine as a component of normal human urine. Partial purification and isolation of D by boronate gel affinity chromatography and reversed-phase HPLC preceded its characterization as a TMS derivative by combined GC/MS techniques. Structure proof is based upon a comparison of mass spectral and chromatographic features of the urinary component to that of an authentic reference sample. Additional data derived from high resolution mass measurements and deuterium isotope-labeling experiments provide confirmation of fragment ion structure. EXPERIMENTAL

A4ateriuls. Methanol (HPLC grade), ammonium acetate buffer, and formic acid were purchased from Wako

Urine collection and chromatographic isolation. A 188-ml aliquot of pooled normal urine obtained from more than 300 normal male and female subjects at the PL Osaka Health Control Center was centrifuged to remove particulate matter and stored at -20°C prior to analysis. The boronate gel affinity chromatography methods used to isolate the urinary nucleosides were a modification of a published procedure (31). Affi-Gel601 (Bio-Rad Labs, Richmond, CA) possessing a specific affinity for cis-hydroxyl groups was packed in a plastic column (60 X 9 mm id.; 1.66 ml bed volume), equilibrated with 20 ml of 0.25 M ammonium acetate (pH 8.8), and washed with 20 ml of 0.1 M formic acid. A 20-ml aliquot of urine was adjusted to pH 8.8 with 4 ml of 2.5 M ammonium acetate (pH 9.5) and loaded onto the column. The column was then washed with 10 ml of 0.25 M ammonium acetate and the nucleosides were eluted with 7 ml of 0.2 M formic acid. The eluate was evaporated under reduced pressure and redissolved in 1 ml of water for HPLC purification. A Shimadzu HPLC system (Shimadzu, Kyoto, Japan) consisting of an LC-6A controller and dual pumps, a SPD-6AV uv-vis detector (260 nm), and a CR3A Chromatopac integrator with recorder was used in the isolation of the individual nucleoside fractions. Analytical and preparatory HPLC procedures used a 5-pm Develosil ODS-5 reversed-phase column (250 X 4.6 mm i.d.; Nomura Chemicals, Nagoya, Japan) and a 15- to 30-pm Develosil ODS-5 precolumn. A filter (1 pm mesh size; Nomura Chemicals) was installed between the injector and the precolumn. Separation of the urinary nucleoside components was achieved using the following solvent gradient (v/v; time in parentheses): water (0 min); 13:87 methanol:water (25 min); 45:55 methanohwater (35 min); water (40 min). The flow rate was 1.1 ml/min with an injection volume of 300 or 350 ~1. The material from each of the fractions was collected and lyophilized for GC/MS analysis. Each lyophilized HPLC fraction was Derivatization. reconstituted in water to a concentration of 1 pg/pl and a lo-p1 aliquot taken to dryness under a stream of nitrogen. The TMS derivatives were prepared by heating each nucleoside fraction in a 50-~1 solution of BSTFA/ TMCS and pyridine (80~20, v/v) for 1 h at 100°C (32). The deuterium-labeled TMS derivatives (33) were similarly prepared using BSTFA-2H1a.

304

REIMER

12

0

FIG. 2. conditions

10

20 Time(min)

HPLC profile of separated and solvent gradient given

14

40

urinary nucleosides. under Experimental.

Column

Muss spectrometry. GC/MS analyses were performed using a Varian Model 3400 gas chromatograph coupled to a Finnigan MAT90 double-focusing (BE) instrument (Finnigan MAT, San Jose, CA). Sample introduction was via splitless injection onto a fused silica DB-5 capillary column (30 m X 0.25 mm; 0.25 pm film thickness; J&W Scientific, Folsom, CA) that was inserted directly into the ion source. The following GC

2.00

AL.

conditions were used initial column temperature, 15O’C; program rate, 6’C/min; final column temperature, 3OO’C (held for 5 min); helium carrier gas at a head pressure of 10 psi; injector port and transfer line temperatures, 300°C. The sample injection volume was 0.5 ~1. The EI mass spectrometry conditions were as follows: ionizing energy, 70 eV; emission current, 1.0 mA; source temperature, 250°C; scan range, m/z 70 to 1000; scan rate, 0.4 s/decade; resolution, 1000 (10% valley definition). High resolution mass measurements on 5,6-dihydrouridine-(TMS)d were carried out at a 5.0 s/decade scan rate and a resolution of 7500; sample introduction was by direct insertion probe and PFK was used for mass calibration.

16

30

ET

RESULTS

AND

DISCUSSION

The HPLC profile of the separated urinary nucleosides is shown in Fig. 2. A total of 30 peaks were fractionated as possible nucleoside components. For several fractions, preliminary identification of the major constituent(s) was made by comparing the HPLC retention times and uv absorption spectra of the urinary components to reference samples. For example, the fraction eluting at 6.63 min (denoted PNU/l; all retention times quoted to the nearest 0.01 min) exhibited an absorbance maximum at 257 nm corresponding to &,,ax of the modi-

1.00

2.00

tb)

mlA

-1.00

\

1.00

: 2 fl

0.50

.0.50

0.00

0.00

1

0.00 250

Wavoloagth FIG. 3. The methanol/water

uv absorbance (v/v).

spectra

of (a) urinary

300

(am) nucleoside

Wavalmgth fraction

PNU/l

and

(b) modified

nucleoside

(run) m’A.

Both

spectra

obtained

in 3%

DIHYDROURIDINE

BY

GAS

CHROMATOGRAPHY/MASS

305

SPECTROMETRY

2562

Pt.KVl/A

FIG.

4.

Reconstructed

-

ion chromatogram

for urinary

nucleoside

fied nucleoside mlA (HPLC retention time 6.13 min; see Fig. 3). However, a GC/MS examination of the TMS derivative of PNU/l revealed the presence of several possible nucleosides in addition to mlA. The reconstructed ion chromatogram (RIC) for PNU/l is shown in Fig. 4. Close inspection of the mass spectra corresponding to the major peaks in the RIC indicated an apparent nucleoside having a GC retention time of 21.73 min (scan 2104). This unknown was given the designation PNU/ l/A for identification purposes. Other suspected nucleosides in PNU/l are indicated in Fig. 4, as are mlA(TMS& and mlA-(TMS)J. No potential nucleosides were detected outside of the displayed scan range. The 70-eV EI mass spectrum of PNU/l/A is shown in Fig. 5a. As with most mass spectral investigations involving nucleosides and their analogs, the main fragment ions can be divided into three groups: (i) those arising from simple losses from the molecular ion, [Ml+, (ii) fragments derived solely from the sugar (S), and (iii) ions containing the intact base (B) plus a portion of the sugar. Structural assignments and mechanistic origins for most of these ions have been described (34-36). Molecular weight information was obtained by examining the ]M]+ and ]M-15]+ regions, the latter ion formed via the loss of a methyl radical. Although a molecular ion was not observed, the molecular weight of the

fraction

PNLJ/l.

Suspected

nucleosides

are denoted

by *.

sample was assigned as 534 on the basis of a [M-CHJ+ ion at m/z 519 (2.7% RI). Other molecular weight-related ions detected were: [M-CHzOTMS]+ (m/z 431, 0.6%), [M-CHs-TMSOH]’ (m/z 429, O-2%), and [M(TMSOH)z]+ (m/z 354,0.7%). Ions normally associated with a ribose-(TMS)z sugar were observed, namely [S-H]+ (m/z 348,0.6%), [S-TMSOH]+ (m/z 259, l-6%), [S-H-CHzOTMS]+ (m/z 245, 2.5%), [S-0-TMSOH]’ (m/z 243, 2.9%), [CdHdOz(TMS)J’ (m/z 230, 4.7%), and [CsHsOz(TMS)J’ (m/z 217,100%). The intense m/z 217 peak and the low abundances of the other sugar-related ions appeared consistent with a base-modified uridine derivative; the mass spectra of this nucleoside subgroup are characterized by intense [C~H~Oz(TMS)z]’ ions, especially compared to m/z 230 ([CdHdOz(TMS)J’). The mass spectra of TMSderivatized pseudouridine (37), 1-methylpseudouridine (38), 5,6-dihydrouridine (37), ribosylthymine (37), and 3-methyluridine (39) support this trend. Subtraction of the mass of the ribose (349 Da) from the suspected molecular mass of the unknown (534 Da) gave an aglycone mass of 185 Da, suggesting a maximum incorporation of one TMS group in the base. Analysis of the TMS-‘Hs derivative of PNU/l/A (see Table 1) confirmed the presence of three TMS groups in the sugar and one in the base. The assignment of 185 Da for the

‘SE+@5

217

-1 .5

80

60 -1 .0 %Rl

40

20

258

300

350

4ee

4%

sea

55e

-0.0 6 e

WI x1e

I

!17

*Et05

4 .4

80,

-1 . 0

%Rl

-0.6

40

3,e 1

a.4

I

20

147

l52@

229

3;s

1269

I

341

I 373

447

a.2

-0.0 0 FIG. dine. 306

5. Low resolution 70 eV mass spectra Background ions are denoted by *.

of the TMS

derivatives

of (a) urinary

unknown

PNU/l/A

and (b) reference

sample

5,6-dihydrouri

DIHYDROURIDINE TABLE

BY

GAS

CHROMATOGRAPHY/MASS

in GC retention times and mass spectra between the reference compound and the PNU/l/A, the identity of the unknown urinary component was established as 5,6-dihydrouridine. Several features of the mass spectrum of 5,6-dihydrouridine-(TMS)d warrant further discussion. In determining the important base- and sugar-related species, two pairs of ions were found to be isobaric: [S-O-TMSOH]+ (S-106) and [B + TMS-CHJ’ (B + 58) at rrz/.z 243 and [S-TMSOH]+ (S-90) and [B + H + TMS]’ (B + 74) at m/z 259. The identities of these fragments were established with the aid of TMS-‘Hg and high resolution measurements (Tables 1 and 2, respectively). For the m/z 243 pairing, the TMS-‘Hg results showed a mass shift of 15 Da (m/z 243 to m/z 258), indicating incorporation of two TMS groups less a TMS-based methyl group. This shift was consistent with the base-related structure [B + TMS-CHJ+, but not with the sugar species [S-0-TMSOH]‘, where a mass shift to m/z 261 (18 Da; two intact TMS groups) would be expected. The measured exact mass of this ion (243.0940) corresponded to an elemental composition of C~H1sN&SizOz (calcd 243.0985), thereby confirming the [B + TMSCHJ’ (B + 58) assignment. Unfortunately, differentiation of the m/z 259 pair was not possible using the TMS‘Hg data as the observed mass shift of 18 Da (m/z 259 to m/z 277) was consistent with both proposed ion structures. However, high resolution mass measurements (259.1190 observed) indicated an elemental composition larities

1

Significant Ions in the Mass Spectra of the TMS and TMS-2Hs Derivatives of the Urinary Unknown PNU/l/A

42 U’MSl

42 (TMS-‘Hs)

Mass shift UW

519 444 431 373 354 348 341 317 301 287 285 259

552 471 458 400 372 375 359 335 319 305 300 277

33 27 27 27 18 27 18 18 18 18 15 18

245 243 230 217 215 171 147 129 103

263 258 248 235 224 177 162 135 112

18 15 18 18 9 6 15 6 9

R See text

for a more

detailed

explanation

307

SPECTROMETRY

No. of TMS gro”ps indicated 4 t-C&) 3 3 3 2 3 2 2 2 2 2 C-(W) 2 2

2 C-C&) 2 2 1 1 t-(X) 2 (-CHa) 1 (-CHs) 1

Structural assignmenta M-15 M-90 M-103 B + 188 M-180 S-1 M-193 B + 132 B + 116 B + 102 B+lOO B + 74 s-90 s-104 B + 58 s-119 S-132 B + 30 B-14 s-202 s-220 S-246

of ion structures.

of the aglycone was substantiated by other ions commonly associated with a pyrimidine base: [B + CzHzO(TMS)z]+ (m/z 373,0.8%), [B + CzHsOzTMS]+ (m/z 317,0.4%), [B + CzHsOTMS]+ (m/z 301,1.9%), [B + CHOTMS]+ (m/z 287,0.9%), [B + CzHzOTMS-CHJ+ (m/z 285, 0.9%), [B + H + TMS]+ (m/z 259, 1.6%), [B + TMS-CHJ+ (nz/z 243,2.9%), [B + H + CHO]+ (m/z 215, 4.6%), [B + CH]’ (m/z 198, l.l%), and [B + HCHJ+ (m/z 171,4.1%). Subtraction of 72 Da, the mass of one TMS group less a hydrogen, from 185 Da gave 113 Da as the mass of the free base, which was indicative of 5,6-dihydrouracil. Comparison of the mass spectrum of PNU/l/A with a library spectrum of 5,6-dihydrouridine-(TMS)k (37) resulted in a close match. Final confirmation of identity was obtained by comparing the GC and mass spectral behavior of PNU/l/A with that of an authentic reference sample of 5,6-dihydrouridine-(TMS)*. The GC retention time for the urinary component was 21.73 min while the reference compound had a retention time of 21.32 min (1.9% error). The low resolution EI mass spectrum of D-(TMS)d (Fig. 5b) exhibited fragmentation and ion intensities essentially identical to its urinary counterpart (Fig. 5a).4 Therefore, on the basis of strong simiweight

’ Careful inspection of the m/z 355 peak is an artifact. The expected m/z 354 (M-180)

in Fig. 5b indicates ion is also present.

that

it

TABLE

2

Elemental Composition and Structural Assignments for Significant Ions in the High Resolution Mass Spectrum of 5,6Dihydrouridine-(TMS)d m/z

(observed) 519.2218 447.1807 373.1766 354.1439 341.1367 317.1359 301.1389 287.1287 259.1190 245.1075 243.0940 230.1165 217.1094 215.0866 198.0794 171.0598 147.0658 129.0403 103.0565

Error’

(mmu) -2.0 -0.4 +3.3 -0.8 -1.4 -0.6 +1.5 -3.1 -0.5 -4.5 +4.5 -0.7 -1.4 -1.4 +3.0 -0.8 +0.4 -3.2 +1.5

Composition

Assignment’

CzOHdzNzSidOe G~JA5~~%06 G5&~2~W3 C~~HzsNzSizOJ G4b5N%04 G&W%O., CizHzsNzSizOs CnHz~NzSizOz G~fb3SLOa G”&Sb% CsHisNzSi~Oz CiOHzzSizOz CsHzLSizOz

CJI~~N2Si03 CsHi4NzSiOz CsHi,NzSiOz C~H~~SizO CsHsSiOz CdHllSiO

’ Error = calculated mass - observed mass; mmu, millimass ’ See text for a more detailed explanation of ion structures. ’ [M-CHs] + ion for lower derivative D-(TMS)s.

M-15 M-15’ B + 188 M-180 M-193 B + 132 B+ll6 B + 102 s-90 s-104 B + 58 s-119 S-132 B + 30 B -t 13 B-14 s-202 s-220 S-246 units.

308

REIMER

of CuH&SizO~ (calcd 259.1185), matching the suggested sugar structure [S-TMSOHJ+ (S-90). No peak for the base-related ion was detected. The ion at m/z 341 (0.9%) in the mass spectra of both the urinary and the reference samples of D-(TMS)b appears to be unique. The TMS-‘Hg spectrum shows two TMS groups (18 Da mass shift), while the measured exact mass (341.1367) indicates an elemental composition of CI~H~~N&O~ (calcd 341.1353). This ion has been tentatively assigned the structure [M-CHs(OTMS)J+ (M-193). The m/z 447 peak appearing in both mass spectra was attributed to the [M-CHJ’ ion arising from the lower derivative 5,6-dihydrouridine-(TMS)a; exact mass measurement confirmed this assignment (see Table 2).

ET

10. Tseng, W.-C., Medina, D., and Randerath, K. (1978) Cuncer Res. 38,1250-1257. 11. de Bruijn, E. A., Driessen, O., van den Bosch, N., and van Strijen, E. (1983) J. Chromutogr. 278,283-289. 12. Aubert, C., Sommadossi, J. R., Coassolo, P., and Cano, J. P. (1982) Biomed. Muss Spectrom. 9,336-339. 13. Mandel, L. R., Srinivasan, P. R., and Borek, E. (1966) Nuture (London) 209,586-588. 14. Tworek, H. A., Bolanowska, W., Bhargava, A. K., Rachlin, E. M., and Chheda, G. B. (1986) Nucleosides Nucleotides 5, 253-263. 15. Trewyn, R. W., Glaser, R., Kelly, D. R., Jackson, D. G., Graham, W. P., and Speicher, C. E. (1982) Cancer 49,2513-2517. 16. Heldman, D. A., Grever, M. R., Speicher, C. E., andTrewyn, R. W. (1983) J. Lub. Clin. Med. 101,783-792. 17. Heldman, D. A., Grever, M. R., Miser, J. S., and Trewyn, R. W. (1983) J. N&l. CuncerInst. 71,269-273. 18. Heldman, D. A., Grever, M. R., and Trewyn, R. W. (1983) BZood

61,291-296.

CONCLUSIONS

The identification of modified nucleosides in human urine by conventional HPLC methods is hindered by the poor response characteristics of the commonly used uv detectors. The analysis of 5,6-dihydrouridine in this report provides an excellent case in point. Lacking a suitable uv chromophore at the standard operating wavelength of 260 nm, D went undetected in the initial HPLC fractionation. However, subsequent analysis by gas chromatography/mass spectrometry provided an excellent means of monitoring the presence of D; the detector (i.e., the mass spectrometer) detected ions of 5,6-dihydrouridine, rather than responding to a general physical property as was the case with HPLC/uv analysis. High detectability, combined with the excellent sensitivity and wealth of structural information available through mass spectrometry, makes GC/MS an excellent instrumental method for the analysis of urinary nucleosides.

19. Gehrke, C. W., Kuo, K. C., Waalkes, T. P., and Borek, E, (1979) Cuncer Res. 39,1150-1153. 20. Speer, J., Gehrke, C. W., Kuo, K. C., Waalkes, T. P., and Borek, E. (1979) Cuncer 44,2120-2123. 21. Borek, E., Sharma, 0. K., and Waalkes, T. P. (1983) Recent Results Cuncer Res. 84,301-316. 22. Borek, E., Waalkes, T. P., and Gehrke, C. W. (1983) Cuncer Detect. Prev. 6.67-71. 23. Fischbein, A., Sharma, 0. K., Selikoff, I. J., and Borek, E. (1983) Cuncer Res. 43,2971-2974. 24. Salvatore, F., Colonna, A., Costanzo, F., Russo, T., Esposito, F., and Cimino, F. (1983) Recent Results Cuncer Res. 84,360-377. 25. Vreken, P., and Tavenier, P. (1987) Ann. Clin. Biochem. 24,59&603. 26. Vold, B. S., Kraus, L. E., Rimer, V. G., and Coombes, R. C. (1986) Cuncer Res. 46,3164-3167. 27. Chheda, G. B., Dutta, S. P., Mittelman, A., Montgomery, J. A., Sethi, S. K., McCloskey, J. A., and Patrzyc, H. B. (1985) Cuneer Res. 45,5958-5963. 28. Chheda, G. B., Patrzyc, H. B., Bhargava, A. K., Crain, P. F., Sethi, S. K., McCloskey, J. A., and Dutta, S. P. (1987) Nucleosides Nucleotides 6,597-611. 29. Chheda, G. B., Tworek, H. A., Bhargava, S. P., and Patrzyc, H. B. (1988) Nucleosides

ACKNOWLEDGMENTS The authors thank Ms. Qingmei their assistance in sample preparation tenance of the mass spectrometer.

Weng and Mr. Peter and in the operation

Baker for and main-

J. T., and

Holley,

R. W. (1965)

B~ochenz.

Bio@zys.

Res.

l&153-157. S. (1978) in Transfer RNA (Altman, S., Ed.), Press, Cambridge, MA. G. (1982) Recent ResuZts Cuncer Res. 84,15-46.

4. Davis, D. R., Griffey, Poulter, C. D. (1986)

R. H., Yamaiznmi, Z., Nishimura, J. Biol. Chem. 26 1,3584-3587.

pp. 16%

33. 34.

S., and

35.

5. Cerutti, P., and Miller, N. (1967) J. Mol. Biol. 26,55-66. 6. Sundaralingham, M., Rao, S. T., and Abola, J. (1971) Science 172,725-727. 7. Rich, A., and RajBhandary, U. L. (1976) Annu. Reu. Biochem. 45, 805-860. 8. Woo, N. H., Roe, B. A., and Rich, A. (1980) Nuture f’London) 286, 346-351.

36.

9. Raba, M., Limburg, K., Burghagen, M,, Katze, J. R., Simsek, Heckman, J. E., RajBhandary, U. L., and Gross, H. J. (1979) J. Biochem. 97,305-318.

M., Eur.

A. K., Rachlin, E., Dutta, Nucleotides 7,417-429.

30. McClure, T. D., Schram, K. H., Nakano, K., and Yasaka, T. (1989) Nucleosides Nucleotides, in press. 31. Nakano, K., Shindo, K., Yasaka, T., and Yamamoto, H. (1985) J. Chromutogr. 332,127-137. 32.

REFERENCES 1. Madison, Commun. 2. Nishimura, 195, MIT 3. Dirheimer,

AL.

3’7.

Schram, K. H., and McCloskey, J. A. (1979) in GLC and HPLC Determination of Therapeutic Agents (Tsuji, K., Ed.), Part 3, pp. 1149-1190, Dekker, New York. McCloskey, J. A., Stillwell, R. N., and Lawson, A. M. (1968) Anul. Chem. 40,233-236. McCloskey, J. A. (1974) in Basic Principles in Nucleic Acid Chemistry (Ts’o, P.O.P., Ed.), Vol 1, pp. 209309, Academic Press, New York. Pang, H., Schram, K. H., Smith, D. L., Gupta, S. P., Townsend, L. B., and McCloskey, J. A. (1982) J. Org. Chem. 47,3923-3932. Reimer, M. L. J., McClure, T. D., and Schram, K. H. (1989) Biomed. Environ. Mass Spectrom. 17,533-542. Basile, B., Scott, M. F., Hsu, F. F., and McCloskey, J. A. (1981) Mass Spectra of Bases, Nucleosides, Nucleotides and Their Derivatives, Univ. of Utah Press, Salt Lake City.

38. Pang, H., Ihara, M., Kuchino, Y., Nishimura, S., Gupta, R., Woese, C. R., and McCloskey, J. A. (1982) J. Biol. Chem. 257, 3589-3592. 39. McClure, T. D., Schram, K. H., Nakano, K., and Yasaka, T. (1989) Nuckosides Nucleotides, in press.