Anal. Chem. 1988, 60, 2719-2722 Kuel, J. C.; Markbs, K. E.; Lee, M. L. H1C CC,J . Hygh Resolut. chrometw. chrometw. Camwr. 1987, 70, 257-262. Fields. S. M.; Markides, K. E.; Lee, M. L. Anal. Chem. 1988, 60, 802-808. FJek)sted, J. C.; Richter. 8. E.; Jackson, W. P.; Lee, M. L. J . Chromatw. 1983, 279, 423-430. Jlnno, K. chrometopaphk 1987. 2 3 , 55-82. CLIfflths. P. R.; P e n t m y , S. L., Jr.; Gbrgenl. A.; Shafer, K. H. Ana/. Chem. 1986, 5 8 , 1349A-1388A. W s i k , S. V.; French, S. B.; Novotny, M. Anal. Chem. 1988, 5 8 , 2258-2258. Monissey, M. A.; HIII, H. H., Jr. Northwest Regional ACS Conference, June 1985. Smith, R. D.; Fellx, W. D.; Fjeldsted, J. C.; Lee. M. L. Anal. Chem. 1882, 5 4 , 1883-1885. Smith, R. D.; Kallnoskl, H. 1.; Udseth, H. R.; Wright, B. W. Anal. Chem. 1884, 5 6 , 2476-2480. Wright, B. W.; Kallnoskl, H. 1.; Udseth, H. R.; Smkh, R. D. !+?C CC.J . High Resolut Chromatogr Chromatogr. Commun 1986, 9 , 145- 153. Kallnoskl, H. T.; Wseth, H. R.; Wrlght, B. W.; Smith, R. D. A M I . Chem. 1986, 58, 2421-2425. Lee, E. D,; Henion, J. D. M C CC, J . Hlgh Resolut. Chromatogr. chromatogr. Commun. 1988, 9 , 172-174. Cousin, J.; Arplno, P. J. J . chrometoy. 1987. 398. 115-141. Kallnoskl, H. 1.; Wseth, H. R.; Chess, E. K.; Smith, R. D. J . chromet q . 1987, 394, 3-14. Smlth, R. D.; Wseth, H. R. Anal. Chem. 1987. 5 9 , 13-22. Lee, E. D.; Henlon, J. D. Anal. chem.1987, 5 9 , 1309-1312. La&. P. A., Jr.; Pentoney, S. L.; oriffiths, P. R.; Wlklns, C. L. Anal. Chem. 1987, 5 9 , 2283-2288. Matsumoto, K.; Tsuge, S.; Hkata, Y. Anal. Chem. 1988, 2 , 3-7. Crowther, J. 8.; Henion, J. D. Anal. Chem. 1985, 5 7 , 2711-2718. Berry, A. J.; Games, D. E.; Perkins. J. R. J . C k o m a t w . 1986, 363, 147-158. Huang, E. C.: Jackson, B. J.; Markides. K. E.; Lee, M. L. chrometographle 1988, 2 5 , 51. Chester, T. L. J . U w o m a t w . 1984, 299, 424-431. Elndf. N.: Munaon. B. Int. J . Smctrom. Ionphvs. 1872. 9 . 141-160.
.
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(29) Soli, J.; Kemmerllng, M.; Schub. 0. Arch. B h h e m . BIophys. 1980, 204. 544-550. (30) W e n , M. W., R J Reynolds Tobacco Co., Winston-Salem. NC, personal communlcatbn. 1987. (31) sheen, S. J.; b v i S , D. L.; DeJone, D. W.; Chaph, J. F. J . A@. Focd Chem. 1978, 26. 259-282. (32) Prenzel, U.; Llchtenthaler, H. K. J . chromatcgr. 1982, 242. 9-19. (33) Wooten, J. B. J . A@. FocdChem. 1985. 3 3 , 419-425. (34) Novotny, M.; Wlesler, D. Seperetlon Methods: New comprehensive Blochemlsby; Deyt, E. Ed.; Elsevler: New York. 1984; Vol. 8, p 41. (35) Kirkland, J. J.; De Stefano, J. J. J . C h o m a t w . Scl. 1970, 8 , 309-314. (38) Walner, I.W. Liq& Ctvomtogephy In phemceut!.cal Development; Aster Publ. Co.,OR 1985; p 239. (37) d e Paults. 1.; K m r , Y.; Johansson, L.; Mmsby, S.; Hall, H.; allemark. M.; Angeby-Mik, K.; Ogren, S . 4 . J . Med. Chem. 1986, 2 9 , 81-89. (38) Claman, H. N.; Miller, S. D.; Conlon, P. J.; Moorhead. J. W. A d . . Immunol. 1980, 3 0 , 121-157. (39) Kristofferson, A.; Ahlstedt, S.; Enander, I. Int. Arch. Allergy Appl. Immund. 1982, 6 9 , 318-321. (40) Blakley, C. R.; Vestal. M. L. Anal. Chem. 1983, 5 5 , 750-754. (41) Apffel. J. A.; Brinkman. U. A. lh.; Frel, R. W.; Evers, E. A. I.M. Anal. Chem. 1983. 5 5 , 2280-2284. (42) Ovchinnkov, Yu. A.; Ivenov, V. 1. Tebehecton 1875. 37,2177-2209. (43) Tabeta. R.; Saito, H. Blcchmkby 1985. 2 4 , 7898-7702. (44) Liddle, W. K.; Wlllis, T. W.; Tu, A. 1.; Sankaram, M. B.; Devarajan, S.; Easwaran, K. R. K. C b m . phys. Wids 1985, 36, 303-308. (45) ByStrOV, V. F. J . Mol. Stnrct. 1985. 726, 529-548.
RECEIVED for review December 15,1987. Resubmitted May 17, 1988. Accepted September 6, 1988. Major financial support for this work was from the U S . Department of Energy, Contract No. DEFG02-86ER604.45. This work was also supported in part by the Gas Research Institute, Contract No. 5084-260-1129, and by Finnigan-MAT. Any opinions, fin-,
Analysis of High Molecular Weight Samples on a Double-Focusing Magnetic Sector Instrument by Supercritical Fluid Chromatography/Mass Spectrometry Vernon N. Reinhold,*Douglas M. Sheeley, Jacob Kuei, and Guor-Rong Her
Department of Nutrition, Harvard School of h b l i c Health, Boston, Massachusetts 021 15
Capillary column supercritical fluid chromatography/mass spectrometry (SFCIMS) has been demonstrated for derivatized oilgosaccharldes in excess of 5 kDa. Columns were prepared with integral pressure restrlctors and directly coupied to a chemical ionization chamber without sklmmers or other Intervening devices. The Interface was maintained at the column temperature with slightly hlgher temperatures at the column tip (ca. 200 "C). The SFC moMie phase was carbon dioxide with ammonia Introduced separately In the chemical ionization chamber to generate (M NH,)' ions. Samples were dedvatited to maintain moblie phase miscC bility, and sample elutlon was provided by a programmed Increase In denslty. Under these conditions the total Ion current could be attributed primarily to molecular-weight-related Ions, and these plots showed no losses in chromatographic fidelity when compared to those for SFC with flame Ionization detection. The current interface provided a slgnal to noise ratio of 1O:l for a 3-ng sample injection.
+
The introduction of fast atom bombardment mass spec0003-2700/88/036O-2719$01.50/0
trometry (FAB MS) (1) and its application to the structural characterization of biopolymers has advanced our understanding of these materials considerably. For peptides and small proteins the impact has been most significant on those samples that have undergone posttranslational modifications and are therefore not amenable to standard analytical procedures (2, 3). The application of FAB MS (4-6), and of comparable soft ionization techniques (e.g., direct chemical ionization (DCI) (7)and laser desorption (LD) (8-10)), has introduced the first hope of a sensitive method for studying carbohydrate structures. Unfortunately, for those techniques requiring a desorption matrix (FAB),a problem arises in the biased presentation of sample components, and it is not uncommon for selected samples to exhibit very poor sensitivity or not work a t all, (e.g., sample suppression). Suppression appears to be less of a problem with the use of a continuous flow FAB probe where the dynamic motion of the surface minimizes this phenomenon (11). For DCI MS, samples are always detected, but heat-initiated desorption leads to pyrrolysis of high molecular weight materials and seriously limits high-mass analysis. An alternative technique not requiring an applied matrix or exhibiting sample suppression has been 0 1988 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 60, NO. 24, DECEMBER 15, 1988
FAB desorption from a polyimide belt (12). But this HPLC-belt interface device is expensive, appears to have high-mass limitations, and is technically demanding. The analysis of nonvolatile compounds by supercritical fluid chromatography (SFC) and the extension of this technique to capillary columns (13) presents an attractive alternative to existing LC/MS configurations. Although yet unrealized, most significant is the flexibility provided to chromatographic and chemical ionization (CI) parameters by simple variations in the mobile phase. These factors, combined with the reduced gas loads, ease of solvent elimination, and potential for encompassing electron capture CI sensitivity, will certainly make CIMS detection a major contributor to molecular characterization. Application of SFC to the separation of glycosphingolipids (14), and preliminary results with direct MS injection of samples dissolved in ammonia supercritical fluids (15)piqued our interest in SFC/CIMS and i b application to high molecular weight biopolymers. This approach, combined with the possibility of obtaining greater structural detail from collision spectra and MS/MS analysis (16),may well circumvent the complications experienced with matrix desorption techniques. This report describes a simple and direct SFC/CIMS interface applicable to derivatized oligosaccharides.
EXPERIMENTAL SECTION Instrumentation. SFC separations were performed on a Model 501p supercritical fluid chromatograph (Lee Scientific, Inc., Salt Lake City, UT), equipped with a flame ionization detector (FID) and a pneumatically actuated submicroliter (0.2 pL) internal loop injection valve (Valco Instrument Co., Inc., Houston, TX). The pneumatically actuated inlet valve provided a split ratio of approximately 1to 5 and sample inlet duration times of 0.01-0.05 min. Approximately 2-10-ng samples were injected for each chromatographic run. Carbon dioxide, SFC grade, (Scott Gases, Plumsteadville, PA) was used as the mobile phase and maintained between 90 and 120 "C. The pump cylinder and injection valve temperatures were held at 0 "C. Columns (10 m X 50 pm id.) were coated with DB-5 (0.2-pm f i i thickness)and obtained from J & W Scientific, Inc., (Folsom, CA). Mobile phase pressure and flow were maintained by fabrication of a flow restrictor similar to that previously reported (17). The detector temperature was held at 390 "C. Chemical ionization (CI) MS was performed on a VG ZAB-SE instrument (VG Instruments, Manchester, U.K.) operating at 8 kV. Ammonia was used as a reagent gas. The ion source was modified for dedicated CI operation by removing the collimating magnets, blocking all inlet ports, and using a narrower exit slit. The SFC column tip was introduced through the single inlet and heated at the very tip with a resistance wire connected to, and controlled by, the DCI unit (see Figure 1). With the exception of this restrictor tip heating (approximately 1 mm) all other temperatures were maintained equal to that of the SFC oven, 120 "C. Samples. Glucose oligomers were obtained from corn syrup by ethanol precipitation or from V-Labs (maltdextrins, MD-6-X, Covington, LA). 0-Cyclodextran was purchased from Aldrich Chemical Co. (Milwaukee, WI). Trimethylchlorosilane was obtained from Regis Chemical Co. (Mortin Grove, IL). Trimethylsilylation was carried out by using an equal mixture of the reagent and pyridine, with warming for 2 h at 65 OC. Samples were permethylated according the procedures described by Ciucanu and Kerek (18). RESULTS AND DISCUSSION Many reports have previously described SFC/MS interfaces (19-28), and a review has recently appeared (29). Specific details have been provided for SFC/MS using chemical ionization (25-27), SFC pressure programming (22, 23), and mobile phase modifiers (28). Most of these studies have involved low-voltage quadrupole mass spectrometers and considerable interface design modifications. From these reports it was not always clear if there were high-mass limita-
I
Capillary Column
i
Contacts for DCI Circuit
Figure 1. Diagram of the SFC/MS probe. The standard DCI probe tip is configured with additional ceramic feedthrough for the SFC column. Contact points provkle electrical coupllng to tabs on the side of the ion block for temperature control. The enlarged tip shows column feedthrough with capillary and resistance wire for tlp heating.
tions. A most interesting report (30) has recently described SFC/CIMS for TMS-derivatized oligosaccharides. This account detected (M + NHJ+ ions up to a limit of DP 7 (degree of polymerization) (m/z 2830), which reflected an instrumental limitation of the quadrupole. In this report, with the use of a tapered restrictor, high interface and ion source temperatures were reported to be necessary, (350 and 300 "C, respectively). This is somewhat disturbing when one is considering future applications to compounds showing greater heat lability and suggests sample volatility may be necessary for restrictor transmission. As an extension of this study we wished to evaluate two major points by using a high-voltage magnetic instrument: (i) were there mass limitations to sample desolvation and ionization and (ii) what were the temperature requirements for the interface and restrictor. Imposed on these primary concerns were additional points that for successful application to biopolymers, both processes should be demonstrated on thermally labile and high molecular weight samples at sensitivities comparable, a t least, to that realized by FAB. Several restrictors have been considered and briefly evaluated for SFC/MS applications. The porous (multipath) frit (31), long drawn capillary (32), laser-drilled disk (15), and integral (17)restrictors have been fitted to SFC columns for MS interfacing studies. The abrupt decompression that would occur with the latter two restrictors appeared better suited for high molecular weight analysis, and more work was focused on these devices. For the other restrictors, we were concerned that elongation of the restrictor zone would provide a corresponding reduction in mobile phase density, introduce subcritical conditions, and allow biopolymer precipitation. For heat-stable analytes, this may be overcome by interface heating where successful sample transmission depends on its volatility. The requirement of restrictor temperatures considerably higher than that required for SFC may be an indication of this condition. This approach abandons the advantages provided by the supercritical fluid mobile phase and can be self-limiting for thermally labile compounds. Although the laser-drilled disks were ideal in principle, they proved to be quite fragile and difficult to align, and required gaskets and a cumbersome mounting device (15). Thus, subsequent work was focused on the integral restrictor which was fabricated directly on the column tip. A diagram of this SFC/MS probe tip is presented in Figure 1. Restrictor tip heating was necessary and was supplied to the last millimeter of column. The precise temperature at the restrictor tip has been difficult to assess because of the ex-
(In,
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ANALYTICAL CHEMISTRY, VOL. 60, NO. 24, DECEMBER 15, 1988 100,
1
I
I
I1
40
20 Time (min)
30
60
90
Flgure 2. Total ionization plot of permethylated glucose polymers. Insert shows SFC/FID chromatogram of the same sample. The split Injection ratio was 5:l.
tremely small area of the heated region. The platinum wire temperature was estimated to be 280 "C on the basis of its length and resistance and the current supplied by the DCI control unit. Lower currents resulted in tip plugging, and the operating current was set slightly above this point. From this temperature, over a rather broad range, no discernible effect on the eluting spectra was detected, although excessive heating currents were not evaluated. The temperature experienced by the eluting analytes must be somewhat lower than that calculated due to their distance from the wire and the cooling contributed by mobile phase evaporation. The larger ceramic cone drawn in Figure 1 is the standard DCI probe modified with an additional ceramic feed to accept the SFC capillary column. As an SFC mobile phase, carbon dioxide has many advantages, but it lacks adequate polarity (at the usual operating densities) to solubilize biopolymers. To ensure mobile phase miscibility and proper column partitioning, we have derivatized all samples. This is consistent with oligosaccharide sequence determination, which requires sample permethylation for the determination of linkage position. Characterization of these products can now be followed by SFC/MS. Shown in Figure 2 is the total ionization profile (TIP) for a series of permethylated glucose homopolymers. The column temperature was maintained a t 90 "C and the samples eluted with pressure programming from 100 to 405 atm at a rate of 5 atm/min. A comparison of this profiie with a separate SFC plot, obtained by using flame ionization detection (FID), (see insert, Figure 2), indicates that the proceses of desolvation and ionization do not disrupt chromatographic fidelity. Further comparison of the TIP (molar detection) and the peak intensity by FID (mass detection) suggests that the ionization efficiency between low and high mass remains constant. It would be interesting to compare the total ions generated from samples of equimolar solutions as one progresses from low to high mass. The slope of the line may provide clues for the efficiency of high-mass desolvation, and ionization. Under the conditions chosen for SFC, the chromatographic resolution of each oligomer decreases with increasing molecular weight, and this loss in selectivity can be observed in both profiles. Selected ion, (M NH4)+,profiles provide a molecular weight distribution of each component in the mixture (Figure 3), and these ions are the major contributors to the total ion current. This fact can be illustrated from the mass spectra of D P 4, DP 7, and DP 10 (Figure 4). To assess this SFC/MS interface and the effectiveness of desolvation and ionization at higher mass, a comparable glucose homopolymer was trimethylsilylated. This derivative would increase each monomer residue by 184 Da as well as provide a sample miscible with supercritical carbon dioxide.
+
60
80
Time (min)
Flgure 3. Selected ion fragmentogram for permethylated glucose polymers, DP 2-DP IO.
149
.ts
91
I,,,,,,,,
o
(M+NH4)+
3 100,
DP 7
, 2104 (M+NH4)+
,
,,
DP 10
I 0
111
I
I000
1
2000
3000
MIZ
Flgure 4. Selected mass spectra of permethylated glucose polymers. DP-2
I
1001
Scan No.
150
300
450
Time (min)
25
50
75
Figure 5. Total ionlzation plot of pertrimethylsllylatedglucose polymers.
This sample has been prepared earlier and chromatographed by SFC (32) and more recently by SFC/MS (30). We wished to compare that data, using a tapered restrictor and higher interface temperatures, with this interface design. The total ion profile for this SFC/MS sample is presented in Figure 5. The column was maintained at 90 "C and the samples eluted with pressure programming from 115 to 400 atm a t a rate of 3 atm/min. Most noticeably, this derivative chromatographs with greater efficiency, showing improved anomer separation for the earlier eluting peaks. The efficiency of ionization appears to be maintained, although quantitation of this fact must await better characterized samples. Profiies of individual adduct ions, (M + NH4)+,for this sample are presented in Figure 6, which shows normalized adduct ions from DP 2 to DP 15. The chromatographic fidelity is again very apparent with anomer separation still observable at DP 15. Figure 7
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ANALYTICAL CHEMISTRY, VOL. 60, NO. 24, DECEMBER 15, 1988
8 is the TIP of 100 pmol of cyclodextrin. Panel 8B is the ammonium adduct ion profiie, m/z 144.6 (M + NH4)+,for that sample. T h e limits of detectability appear to approach 2 pmol. A t this concentration no peaks were detected in the total ion profile, although the ion was easily detected at this concentration, (Figure 8C).
25
50
75
Retention t i m e (min)
Figure 6. Selected ion profile of trimethylsilylatedglucose polymers. 5850 (M+NH4)'
100
LITERATURE CITED
I I TO
.
TO
113
-c
MI2
Flgure 7. Mass spectrum of trimethylsilylated glucose oligomer, DP 15.
68!
28
/*Ii 48
o i....
ACKNOWLEDGMENT We gratefully acknowledge the generous and combined support of VG Instruments and Lee Scientific for providing instrumentation. From T. L. Chester and G. D. Owens (Procter & Gamble) and Lee Scientific we acknowledge gifts of tapered and frit restrictors, respectively.
. : , -
. -
..,..
- -
:.
.>:
.,..
+..,,.
/I
FIgm 8. SFC/MS total ionization plot obtained from 15O-ng injection
permethylated &cyclodextrin, (A); plot of molecular ion adduct (M + NH,)' m l z 1446, for above injection, (6); plot of molecular ion adduct (M NH,)' for S n g injection, (C). of
+
is the mass spectrum for DP 15. The overall sensitivity of this interface was studied by use of known concentrations of cyclodextrin that had been permethylated previous to analysis. Shown at the top of Figure
(1) Barber, M.; Bordoll, R. S.; Sedgwlck, R. D.; Tyler, A. N. J. Chem. Soc., Chem. Commun. 1981, 213, 1497-1501. (2) Biemann, K.; Scoble, H. A. Sclence (Washlngton, D . C . ) 1987, 237, 992-998. (3) Blemann, K. Anal. Chem. 1988, 58, A1288-A1305. (4) Reinhold, V. N.; Carr, S. A. Mass Spectrom. Rev. Wag, 2 , 153-221. (5) Reinhold, V. N. I n Mass Spectrometry in Bbmedhl Research; Gaskill, S. J., Ed.; Wliey: New York, 1986; pp 181-213. (6) Dell, A. Adv. Cerbohydr. Chem. Blochem. 1987, 45, 17-72. (7) Reinhold, V. N. OligosacchanbeC h e m h l Ionizatbn, Methods in Enzymology, Complex Caf6ohy&ates, Part €; Glnsburg, V.,Ed.; Academic: New York, 1987; vol. 138, pp 59-84. (8) Laude, D. A., Jr.; Pentoney, S. L., Jr.; Grlffiths, P. R.; Wilklns, C. L. Anal. Chem. 1987, 59, 2283-2288. (9) Lam, 2.; Comlsarow. M. 6.; Dutton, 0.G. S.;Weil, D. A,; Bjarnason, A. Rapld Common. Mass Spectrom. 1987, 1, 83-87. (10) Coates, M. L.; Wllklns, C. L. Anal. Chem. 1887, 59, 197-200. (11) CaDrbli, R. M.: Moore, W. T.: Fan, T. RaDa Commun. Mass S m c trom. W87, 1 , 15-18. (12) Santikarn, S.; Her, G. R.; Relnhold. V. N. Carbohydr. Chem. 1987, 6 , 129-139 .- - .- -. (13) Novotny, M.; Sprlngston, S. R.; Peadon, P. A.; Fjeldsted, J. C.; Lee, M. L. Anal. Chem. 1981, 53, 407A-414A. (14) Kuel, J. C. H.; Her, G. R.; Reinhold, V. N. Anal. Biochem. 1988, 172, 228-234. (15) Santlkarn, S.; Reinhold, V. N. Presented at the 33rd Annual Conference on Mass Spectrometry and Allied Topics, San Dlego, CA, May 26-31, 1985. (16) Carr, S. A.; Reinhold, V. N.; Green, 6. N.; Hass, J. R. Biomed. Mass Spectrom. 1985, 12(6), 288-295. (17) Guthrle, E. J.; Schwartz, H. E. J . Chromatogr. Sci. 1988, 2 4 , 236-240. (18) Clucanu, I.: Kerek. F. Carbohydr. Res. 1984, 131, 209-217. (19) Randall, L. G.; Wahrhaftlg, A. L. Rev. Sci. Instrum. 1981, 52, 1283-1 295. (20) Yonker, C. R.; Wright, B. W.; Udseth, H. R.; Smith, R. D. Ber. BunsenGes. Phys. Chem. 1984, 88, 908-911. (21) Smith, R. D.; Udseth, H. R.; Kalinoskl, H. T. Anal. Chem. 1984, 56, 2971-2974. (22) Smith, R. D.; Kalinoski, H. T.; Udseth, H. R.; Wright, B. W. Anal. Chem. 1884, 56, 2476-2480. (23) Smith, R. D.; Chapman, E. G.; Wright, B. W. Anal. Chem. 1985, 5 7 , 2829-2836. (24) Smith. R. D.; Udseth, H. R. AnalChem. 1987, 59, 13-22. (25) Smith. R. D.: Fjeldsted, J. C.; Lee, M. L. J . Chromatogr. 1982, 247, 231-243. (26) SmMi R. D.; Udseth, H. R. Anal. Chem. 1983, 55, 2266-2272. (27) Wright, B. W.; Udseth, H. R.; Smith, R. D.: Hazlett, R. N. J. Chromatwr. 1984, 314, 253-282. (28) Wright, B. W.; Kalinoski, H. T.; Smith, R. D. Anal. Chem. 1985, 5 7 , 2823-2829. (29) Smith, R. D.; Kalinoskl, H. T.: Udseth, H. R. Msss Spectrom. Rev. 1987, 6 , 445-496. (30) Pinkston, J. D.; Owens, G. D.; Mililngton, D. S.; Burkes, L. J.; Delaney, T. E. Anal. Chem. 1988, 10, 982-966. (31) Markldes, K. E.; Fields, S. M.; Lee, M. L. J . Chromatogr. 1986, 2 4 , 254-257. (32) Chester, T. L.; Innis, D. P. HRC CC, J . H/gh Resolot. Chromatogr. Chromatogr. Commun. 1986, 9 , 178-181.
RECEIVED for review April 7,1988. Accepted September 28, 1988. Support from the Army Medical Research Institute for Infectious Diseases, (DAMD17-85-C-5274);National Institutes of Health, (RR1419); and National Science Foundation (PCM 8300342) are gratefully acknowledged. The views, opinions, and/or findings of the research do not necessarily reflect the position or decision of the United States Army, and no official endorsement should be inferred.
