Mass Spectrometry - American Chemical

Chapter 9

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Qualitative Analysis of Pharmaceuticals by Thermospray Liquid Chromatography/Mass Spectrometry Nemadectins

and

Tetracyclines

Grant B. Kenion,GuyT.Carter,JaweedAshraf,MarshallM.Siegel,and Donald B. Borders Medical Research Division, American Cyanamid Company, Pearl River,NY10965

Our primary use of thermospray liquid chromatography-mass spectrometry (LC/MS) has been as a tool with which to search for novel compounds in fermentation broths. Two classes of compounds for which this technique has been particularly useful are the nemadectins (a family of antiparasitic macrolides recently isolated from Streptomyces cyaneogriseus sp. noncyanogenus (1)) and the tetracyclines. The optimal thermospray LC/MS compatible HPLC separation for the nemadectins alpha, beta, gamma, and lambda consists of a methanol/ water solvent gradient solvent system and a C-18 column. Interpretation of the thermospray mass spectra in positive and negative ion modes using either discharge electrode or filament ionization demonstrate that the electron capture ionization dominated negative ion spectra provide excellent molecular weight information, while the positive ion spectra provide useful structural information due to extensive fragmentation. For the tetracyclines,a thermospray compatible HPLC method using a C-4 column and a solvent system consisting of dimethylformamide and ammonium acetate buffer is capable of separating tetracycline, 6-demethyltetracycline, declomycin, and chlortetracycline. The positive ion thermospray mass spectra of the tetracyclines are simple, with the base peak consisting of the proton adduct of the parent compound. The negative ion thermospray spectra (with the discharge electrode on) display intense, structurally diagnostic fragmentation. 0097-6156/90/0420-0140$07.50/0 © 1990 American Chemical Society Brown; Liquid Chromatography/Mass Spectrometry ACS Symposium Series; American Chemical Society: Washington, DC, 1990.

9. KENION ET AL.

Qualitative Analysis ofPharmaceuticah

141

Nemadectins alpha, beta, gamma, and lambda (1-4) are produced v i a a fermentation process (2.) that a l s o produces a number of novel minor components of s i m i l a r s t r u c t u r e . Our i n i t i a l o b j e c t i v e was t o adapt our HPLC method f o r thermospray LC/MS a n a l y s i s . The second o b j e c t i v e was t o evaluate i o n i z a t i o n and d e t e c t i o n modes to determine the best combination i n terms of s e n s i t i v i t y , molecular weight information, and s t r u c t u r a l l y d i a g n o s t i c fragmentation data. The ultimate goal was t o develop an a n a l y t i c a l method r e t a i n i n g the r e s o l v i n g power of the o r i g i n a l HPLC method (1), with adequate s e n s i t i v i t y t o detect minor components and s u f f i c i e n t fragmentation t o d i f f e r e n t i a t e isomeric compounds. Thermospray mass spectra often provide e x c e l l e n t molecular weight information, with some a d d i t i o n a l s t r u c t u r a l information being provided by fragment ions, v i a an i o n i z a t i o n mechanism s i m i l a r i n "softness" t o chemical i o n i z a t i o n ( CI ) Q ) . Spectra obtained by previous researchers from 22,23 dihydroavermectin Bia/ a macrolide s i m i l a r i n s t r u c t u r e t o the nemadectins, demonstrated that chemical i o n i z a t i o n mass spectrometry(A) and thermospray LC/MS (ϋ) provided e x c e l l e n t r e s u l t s . We a n t i c i p a t e d , therefore, that the nemadectins would a l s o be amenable t o thermospray LC/MS analysis. High performance l i q u i d chromatography of t e t r a c y c l i n e s using reversed phase columns (6-15) has been a subject of intense i n t e r e s t over the l a s t decade and a h a l f . Previous methods have g e n e r a l l y used sodium phosphate (£), ammonium carbonate (12.), c i t r i c a c i d (14.), t e t r a a l k y l ammonium s a l t s (15) or other n o n - v o l a t i l e b u f f e r i n g agents t o improve r e s o l u t i o n , unfortunately, while these b u f f e r s are f i n e when using UV d e t e c t i o n , they are not the b u f f e r s of choice f o r thermospray LC/MS a n a l y s i s . We present a reversed phase HPLC separation that i s thermospray compatible, as shown by p o s i t i v e and negative i o n spectra and a t o t a l i o n chromatogram showing good r e s o l u t i o n between 6-demethyltetracycline, t e t r a c y c l i n e , declomycin, and c h l o r t e t r a c y c l i n e . Our choice of dimethylformamide as the organic modifier was based on e x c e l l e n t s o l u b i l i t y and chromatographic c h a r a c t e r i s t i c s f o r t e t r a c y c l i n e s obtained by R. Leese and F. Barbatschi ( 1 £ ) f o r an HPLC system using a non v o l a t i l e b u f f e r . Ammonium acetate was chosen on the b a s i s of i t s optimal (12) c o m p a t i b i l i t y with thermospray LC/MS a n a l y s i s . I n i t i a l attempts using p e r f l u o r i n a t e d b u f f e r s were plagued by high mass noise (probably from column bleed and c l u t e r formation). I n i t i a l attempts using ammonium acetate and ammonium formate b u f f e r s with C-8 and C-18 columns d i d not provide adequate separation. A r

Brown; Liquid Chromatography/Mass Spectrometry ACS Symposium Series; American Chemical Society: Washington, DC, 1990.

142

LIQUID CHROMATOGRAPHY/MASS

SPECTROMETRY

s o l u t i o n of the four standards (6-demethyltetracycline, t e t r a c y c l i n e , declomycin, and c h l o r t e t r a c y c l i n e ) e l u t e d as a s i n g l e peak with very l i t t l e r e t e n t i o n . A C-4 reversed phase column was the only column we t e s t e d that could provide adequate separation using ammonium acetate or ammonium formate b u f f e r s . MATERIALS AND METHODS Nemadectins A l l mass s p e c t r a l analyses other than p o s i t i v e ion desorption chemical i o n i z a t i o n ( DCI ) mass spectra were performed using a Finnigan ( San Jose, CA ) TSP-46 Dedicated Thermospray LC/MS s i n g l e quadrupole mass spectrometer. Data were acquired using Revision 5.5 of the INCOS data system software. The vaporizer temperature f o r LC/MS a n a l y s i s was set at 95°C, while the j e t temperature was set at 220°C. Optimization of temperature parameters was performed using maximum molecular ion i n t e n s i t y , gaussian peak shape, and maximum t o t a l i o n response as q u a l i t y i n d i c a t o r s . A mass range of 400-650 amu was scanned every 2 seconds. The e l e c t r o n m u l t i p l i e r was set at 1800 v o l t s . When i n use, the discharge electrode was set at 1 KV. The chemical i o n i z a t i o n experiments were performed using the EI/CI source. U l t r a Pure Grade methane purchased from Linde ( Danbury, CT ) was used as the reagent gas. The i o n i z e r temperature was set at 100°C. Source pressure was optimized at 1.44 t o r r . The e l e c t r o n energy was set at 70 eV. The e l e c t r o n m u l t i p l i e r was set at 1300 v o l t s . A mass range of 100-800 amu was scanned every 2 seconds. Samples were introduced v i a a d i r e c t i n s e r t i o n probe heated from 30°C t o 400°C at 120°C / min. (Structurel) A l l p o s i t i v e ion DCI mass spectra were obtained from a Finnigan MAT 90 double focusing magnetic sector mass spectrometer. The e l e c t r o n energy was set at 150 eV. The instrument was set t o scan from 100-850 amu at 2 seconds per decade with a 1 second InterScan time. Ammonia was used as the reagent gas at a source pressure of lxlO~ Torr. A Spectra-Physics ( San Jose, CA ) SP 8800 Ternary Gradient HPLC pumping system was used. Methanol and water of HPLC grade were purchased from J.T. Baker ( P h i l l i p s o n , NJ ) and used without f u r t h e r p u r i f i c a t i o n . I n j e c t i o n of samples f o r LC/MS a n a l y s i s was performed using a Waters ( S p r i n g f i e l d , MA ) WISP A u t o i n j e c t o r . HPLC separations were accomplished with a Perkin Elmer 3X3 CR C-18 column. A solvent gradient of 60% methanol, 40% water to 90% methanol, 10% water i n 30 4


9.

143

Qualitative Analysis ofPharmaceuticals

KENION ET AL.

minutes at a flow rate of 1.2 ml/min provided best r e s u l t s . A t o t a l ion chromatogram from an LC/MS a n a l y s i s i s shown i n Figure 1 f o r a standard s o l u t i o n of ( i n order of e l u t i o n ) nemadectins beta, gamma, alpha, and lambda with a concentration of approximately 1 mg/ml of each analyte. Use of ammonium acetate b u f f e r causes gamma and alpha to coelute, as does using a c e t o n i t r i l e i n s t e a d of methanol as the organic m o d i f i e r . Authentic samples of the nemadectins alpha, beta, gamma, and lambda were obtained as p r e v i o u s l y described (1). Tetracyclines A l l t e t r a c y c l i n e , 6-demethyltetracycline, declomycin, and c h l o r t e t r a c y c l i n e standards were provided by Lederle Laboratories as the hydrochloride s a l t s . A l l f a s t atom bombardment spectra were obtained with a VG (Manchester, UK) ZAB SE equipped with a cesium ion gun operated at 30 kV. The optimum matrix was "magic b u l l e t " with a methanol solvent. (Structure2) A l l thermospray mass spectra were obtained from a Finnigan TSP-46 Dedicated Thermospray LC/MS coupled to a Spectra-Physics SP-8800 Ternary HPLC system. A Rainin Dynamax 150°A 25cm 12mm C-4 column (catalog #83-502-C) with matching C-4 guard colum (catalog # 83-502G) was used throughout. The 12mm p a r t i c l e s i z e i s important, as pressure problems can a r i s e when using dimethylformamide as the organic modifier due to the high v i s c o s i t y of the solvent. The f o l l o w i n g solvent gradient system provides optimum r e s u l t s at a flow rate of 1.2 ml/min: TIME

%0.1

M NH4OAC

(min)

(2%ACN.

pH fi.Fii

0 10 35 40 60

80 80 80 50 50

%H20%

DMF

15 0 0 0 0

5 20 20 50 50

Although a l l of the standards presented i n t h i s manuscript w i l l e l u t e at a solvent composition of 20% dimethylformamide, other members of t h i s c l a s s of compounds w i l l not e l u t e with a solvent composition l e s s than 50% dimethylformamide. The optimum mass spectrometer parameters are as follows : Scan Range: 300-800 amu, 2 seconds scan rate Vaporizer Temperature: 120°C, change to 130°C when DMF composition i s above 20%. Jet Temperature: 250°C Discharge E l e c t r o d e : 1KV


144

LIQUID

CHROMATOGRAPHY/MASS

SPECTROMETRY

OH

R

LLF28249a 1 LLF282493 J LLF28249Y I LLF28249X ?

l

H H Me Me

1pr Me Me ipr

Structure 1

OH

Ο

OH Ο

6-Demethyltetracycline Tetracycline Declomycin Chlortetracycline

Ri=H, R2=H Ri=H, R =CH R!=C1, R =H Ri=Cl, R =CH 2

3

2

2

3

Structure 2



•ι

Figure 1.

Γ""—r~ 100 3:20 300 10:00

400 13:20

500 16:40

600 20:00

L

700 23:20

lambda

Reconstructed ion chromatogram of nemadectins alpha, beta, gamma, and lambda.

200 6:40

,

beta

gamma

alpha

146

LIQUID

CHROMATOGRAPHY/MASS

SPECTROMETRY

I t s h o u l d be n o t e d t h a t a h i g h sample t h r o u g h p u t u s i n g t h i s a n a l y s i s w i l l o f t e n cause t h e quadruple rods t o become c o n t a m i n a t e d . When u s i n g t h i s method, one s h o u l d c h e c k t h e raw d a t a p e r i o d i c a l l y a n d e n s u r e t h a t h i g h mass assignment i s s t a b l e . RESULTS AND DISCUSSION Nemadectins The o p t i m a l HPLC s e p a r a t i o n c o n d i t i o n s f o r t h e nemadectins p r e c l u d e d t h e use o f " b u f f e r o n l y " i o n i z a t i o n . The F i n n i g a n T S P - 4 6 t h e r m o s p r a y s o u r c e i s equipped w i t h a f i l a m e n t and a d i s c h a r g e e l e c t r o d e as c h o i c e s f o r an e x t e r n a l i o n i z a t i o n method. B o t h p o s i t i v e and n e g a t i v e i o n d e t e c t i o n a r e a v a i l a b l e . The b e s t c h o i c e f o r an i o n i z a t i o n method a n d f o r t h e mode o f i o n d e t e c t i o n w o u l d have t o p r o v i d e a d e q u a t e s e n s i t i v i t y f o r m i n o r component a n a l y s i s , unambiguous m o l e c u l a r w e i g h t i n f o r m a t i o n , and s u f f i c i e n t fragmentation t o d i f f e r e n t i a t e between components w i t h s i m i l a r r e t e n t i o n times and i d e n t i c a l molecular weights. E a c h o f t h e s t a n d a r d n e m a d e c t i n s was a n a l y z e d u s i n g f i l a m e n t i o n i z a t i o n i n b o t h p o s i t i v e and n e g a t i v e i o n d e t e c t i o n modes a n d u s i n g t h e d i s c h a r g e e l e c t r o d e i n b o t h d e t e c t i o n modes. T h e s e d a t a were t h e n compared w i t h r e g a r d t o s e n s i t i v i t y , degree o f fragmentation ( and t h e r e s u l t i n g s t r u c t u r a l information content ) , and q u a l i t y o f molecular weight i n f o r m a t i o n . The p o s i t i v e i o n s p e c t r a o b t a i n e d u s i n g f i l a m e n t i o n i z a t i o n (as shown i n F i g u r e 2 a n d i n T a b l e I) a r e c h a r a c t e r i z e d b y an (M+H ) i o n w i t h a r e l a t i v e abundance o f 10-20%. The most abundant f r a g m e n t i o n s o b s e r v e d i n these s p e c t r a correspond t o c o n s e c u t i v e l o s s e s o f water f r o m (M+H) . The s p e c t r a o f t h e gamma a n d lambda components a l s o c o n t a i n i o n s r e s u l t i n g from l o s s o f m e t h a n o l (m/z 531 a n d 567; m/z 577 a n d 595 r e s p e c t i v e l y ) , p r e s u m a b l y a r i s i n g f r o m t h e C-5 methoxy g r o u p . Ions r e s u l t i n g f r o m l o s s o f t h r e e w a t e r m o l e c u l e s f r o m gamma and lambda must r e s u l t f r o m h y d r o l y s i s a t t h e a c e t a l a t C-21 o r t h e e t h e r a t C-6 a n d s u b s e q u e n t l o s s o f w a t e r , a s t h e i n t a c t m o l e c u l e s o n l y have two h y d r o x y g r o u p s e a c h . In a d d i t i o n t o these e l i m i n a t i o n s , t h e r e a r e i o n s d e r i v e d from r e t r o D i e l s - A l d e r fragmentations i n the s p e c t r a . E a c h o f t h e compounds shows an i o n d e r i v e d f r o m t h e r e t r o D i e l s - A l d e r f r a g m e n t a t i o n o f t h e (M-2 H 2 O ) s p e c i e s , f r a g m e n t a t i o n a i n Scheme 1. The masses o f t h e s e i o n s a r e u s e f u l i n d e t e r m i n i n g changes i n t h e s u b s t i t u t i o n p a t t e r n on t h e c o r e p a r t o f t h e m o l e c u l e a p a r t f r o m t h e C-25 s i d e c h a i n . Thus b o t h a l p h a a n d b e t a g i v e r i s e t o m/z 4 65 i o n s , whereas gamma a n d lambda y i e l d +

+

+


9. KENION ET AL.


581 563

479

531

447 f

•' Γ '

Ί

Figure 2. Positive ion spectra using filament ionization. Continued on next pa^e. American unemical Society

Library

1155 16th St., N.W.Spectrometry Brown; Liquid Chromatography/Mass ACS Symposium Series; Washington, American Chemical D.C.Society: 20036Washington, DC, 1990.

147

148

LIQUID CHROMATOGRAPHY/MASS

SPECTROMETRY

58.8-

475

t

ι

. • H • ιί •»• ., • I 480

• ι I

5Q0

Ί

d.

50.0 3?3

338

355 371

381

HMIII|1I

M/Z

320

34o

360

38o

400

420

440

il

46Q

L«2 480

500

Figure 6. Continued. Negative ion spectra of c. Declomycin and d. Chlortetracycline.



460 442 434

390 371

446 428 420

375

-

426

399

356 338

412

386

342 324

loss of water

loss of HC1

loss of (CH3)2N

loss of (CH3)2N and C0NH2

loss of (CH3)2N, water, and C0NH2

loss of HC1 and (CH3)2N

398

424

477

463

443

429

deprotonation

loss of water and HC1

478

464

444

chlortetracycline

430

declomycin

tetracycline

electron capture

6-demethyltetracycline

Table III. Negative ion Thermospray fragments

164

LIQUID CHROMATOGRAPHY/MASS SPECTROMETRY

be r e l i a b l e , due t o s i g n i f i c a n t i r r e v e r s i b l e binding o f some analytes, p a r t i c u l a r l y the c h l o r i n a t e d species, t o the column).The extensive fragmentations o f the t e t r a c y c l i n e s i n the negative ion thermospray mass spectra provide u s e f u l s t r u c t u r a l information. The p o s i t i v e ion mode provides unambiguous molecular weight information f o r the t e t r a c y c l i n e s due t o the intense M+l ions and lack of extensive fragmentation.

Acknowledgments The authors would l i k e t o thank Dr. Richard Leese and Dr. Fred Barbatschi,also of American Cyanamid, f o r h e l p f u l suggestions. Literature Cited 1. 2.

3. 4. 5. 6. 7. 8. 9. 10. 11.

12. 13. 14. 15.

Carter, G.T., Nietsche, J.Α., Hertz, M.R., Williams, D.R., Siegal, M.M., Morton, G.O., James, J.C., Borders, D.B. J.Antibiotics, 1988, 41 , 519. Goodman, J.J., Torrey, M.J., Korshalla, J.Α., Pinho, F.,Testa, R.T. Program and Abstracts of fhe 27th Intersci.Conf.on Antimicrob.Agents Chemother., 1987, p. 271. Blakely, C.M. , Vestal, M.L. Anal. Chem., 1984,55,750. Tway, P.C., Downing, G.V., Slayback, J.R.B., Rahn, G.S. and Isense, R.K. Biomed. Mass Spectrom., 1987 , 11, 172. Gartiez, D.A., Vestal, M.L. LC, 1985,3, 334. Tsuji, Κ.,Robertson, J.H., Beyer, W.F. Anal.Chem., 1974, 46, 539. Tsuji, K.,Robertson, J.H. J.Pharm.Sci., 1975, 110, 103. Knox, J.H., Jurand,J. J.Chromatogr., 1975, 110, 103. Knox, J.H., Pryde, A. J.Chromatogr., 1975, 112, 171. Chevalier, G., Bollet, C., Rohrbach, P., Risse, C., Caude M., Rosset,R. J.Chromatogr., 1976,124, 754. Nilsson-Ehle, I., Yoshikawa, J.T., Schotz, M.C., Guze, L.B. Antimicrob.AgentsChemother., 1976, 9, 754. White, E.R., Carrol, M.A.,Zarembo,J.E. Bender, J.Antibiot., 1975, 28, 205. De Leenher, A.P., Nelis, H.J.C.F. J.Chromatogr., 1977, 140, 293. De Leenher, A.P., Nelis, H.J.C.F. J.Pharm.Sci., 1979, 68, 999. Mourot, D., Delepine, B., Boisseau, J. and Gayot,G. J.Chromatogr., 1980, 190, 486.


9. KENION ET AL. 16.

17. 18. 18. 20.


Leese, R., Barbatschi, F. HPLC Assay For Chlortetracycline in Animal Feed, presented at AOAC National Meeting, Washington, D.C., October, 1983. Voyksner, R.D., Haney, C.A. Anal.Chem., 1985, 57, 991. Kebarle, P. Choudhury, S. Chem. Rev., 1987, 87, 513. Parker, C.E., Smith, R.W., Gaskell, S.J., Bursey, M.M. Anal. Chem., 1986, 58, 1661. Hoffman, D.R.

J.Org.Chem.,

1966, 31,

792.

RECEIVED October 24, 1989


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