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We have found that these salt mixtures age and their FAB spectra will change over time. It is, therefore, necessary to adjust the salt concentrations and the reference table when a new calibration mixture is made. Conclusions There are wide varieties of reference compounds available for use in mass spectrometry and it is impossible in this brief space to give all the details of their use. Table I summarizes the commonly used reference compounds and Appendix 2 at the end of the volume includes tables with accurate masses of reference compound peaks.
[15] C h e m i c a l D e r i v a t i z a t i o n for M a s s S p e c t r o m e t r y
By DANIEL R. KNAPP Introduction Chemical derivatization has played an imporant role in organic mass spectrometry from the earliest practice of the technique.1 Although the major impetus for its early use was to confer the necessary volatility to sample compounds, chemical derivatization was also used to increase the information yield from mass spectral data. With decades of progress in mass spectrometry and the development of a wide range of new techniques, chemical derivatization still plays an important role. This chapter will give an overview of the use of chemical derivatization in mass spectrometry with particular emphasis on analytical strategy aspects. General considerations with respect to the practical aspects of chemical derivatizations and some general methods will also be described. More specific applications are given in later chapters in this volume, as well as in a recent review 2 and monograph. 3 i K. Biemann, "Mass Spectrometry, Organic Chemical Applications." McGraw-Hill, New York, 1962. 2 R. J. Anderegg, Mass Spectrom. Rev. 7, 395 (1988). 3 D. R. Knapp, "Handbook of Analytical Derivatization Reactions." Wiley, New York, 1979.
METHODS IN ENZYMOLOGY, VOL. 193
Copyright © 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
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Reasons for Use of Chemical Derivatization in Mass Spectrometry The reasons for utilizing chemical derivatization in relation to mass spectrometry can be summarized as follows: (1) enhancement of volatility; (2) degradation of the sample molecule to smaller subunits; (3) enhancement of detectability; (4) enhancement of separability; (5) modification of fragmentation: (a) enhancement of molecular weight related ions and (b) enhancement of structurally informative ions; (6) determination of functional groups. Volatility enhancement was a requirement for many samples in the early days of mass spectrometry when the available inlet systems required significant vapor pressure. In current practice, derivatization for volatility enhancement is used primarily in relation to interfaced gas chromatography-mass spectrometry (GC/MS) where such derivatization serves to enable or improve the gas chromatography. Vapor pressure of a compound is influenced by intermolecular attractions due to dispersion (van der Waals) forces, ionic interactions, and hydrogen bonds. The total dispersion forces increase with molecular size and little can be done with respect to these forces to increase volatility other than reduce the size of the molecule (see below). Chemical derivatization for volatility enhancement is aimed at reducing ionic and hydrogen bond interactions by conversion of ionizable groups to nonionizable derivatives (e.g., carboxyl groups to esters); replacing hydrogens bound to heteroatoms (N-H, O-H, S-H) with alkyl, acyl, silyl, or other groups; and reducing the polarity of hydrogen bond accepting groups (e.g., conversion of carbonyls to methoximes). An example of a multistep derivatization to increase volatility is the conversion of peptides (1) to the volatile polyamino alcohol trimethylsilyl ethers (11).4 Not only are these derivatives sufficiently volatile for GC/ MS, but they also have the added advantage of yielding stable, structurally informative ammonium-type ions in the mass spectrum. Thus, these derivatives (as is often the objective) serve a dual purpose. R O I II H=N-(CH-C)n--OH
peptide
1. acylation (R'CO-) R 2. reduction I ~ R'CH=NH-(CH-CH2)n--OTMS 3. trimethylsilylation
(I)
trimethylsilyl (TMS) polyamino alcohol
(ii)
Degradation of sample molecules by chemical means was originally employed for volatility consideration, i.e., large molecules were degraded to smaller constituent molecules with sufficient vapor pressure to be ana4 K. B i e m a n n , this v o l u m e [18].
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GENERAL TECHNIQUES
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lyzed. In current practice, degradations are still used to identify constituent subunits, but molecular size reductions are driven more by instrument mass range limits than volatility. In the case of tandem mass spectrometry, the need for degradation to smaller size fragments may be motivated by molecular size limits of current ion dissociation technology rather than the analyzer mass range. An example of a contemporary use of degradation is the acetolysis of glycoproteins to cleave off the oligosaccharide components as peracetylated carbohydratesS: Glycoprotein
acetolysis
~Peracetylatedoligosaccharides
Derivatization for detectability enhancement involves chemical conversions to promote the formation of stable charged species with either little fragmentation or characteristic fragmentation behavior to yield intense well-defined ions. An example is the attachment of an electrophoric moiety to promote the formation of negative ions i n " negative ion chemical ionization" (NICI) mass spectrometry. This approach has been used, in particular, for eicosanoids for high sensitivity quantitation. 6 For example, prostaglandin E 2 ( m ) is converted in three steps to the methoxime (MO), trimethylsilyl (TMS), pentafluorobenzyl (PFB) derivative (IV) which exhibits good gas chromatographic behavior. Under NICI conditions the derivative captures an electron to yield a negatively charged molecular radical anion which readily loses a pentafluorobenzyl radical to form a relatively stable carboxylate anion. The resulting mass spectrum exhibits an intense ( M - 181)- peak which can be monitored by GC/MS selectedion monitoring for very sensitive, and selective, detection and quantitation of eicosanoids in biological samples. 0 ~ ~ C O O H
1. methoximation 2. pentafluorobenzyl esterification ~ 3. silylation
HO
OH Prostaglandin E2
(no
N-OCH~ \\ ~ " ~ , ,' TMSO
~COOCH=CaFs J
. i OTM$
MO-PFB-TMS Derivative
(iv)
The advent of fast atom bombardment (FAB) ionization 7 has led to the use of chemical derivatization for detectability enhancement by the 5 A. Dell, this volume [35]. 6 I. Blair, this series, Vol. 187, p. 13. 7 In this chapter, the term, FAB, is used generically to include ion bombardment of samples in liquid matrices.
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intentional introduction of a charged group into the molecule, which is just the opposite of the traditional masking of polar groups. For example, the ketosteroid androsterone (V), which is undetectable under normal FAB conditions (glycerol, 4/zg//zl) in the native state, gives an intense molecular ion as the quaternary ammonium derivative (VII) resulting from derivatization with Girard's reagent T (VI). s
0 H
O +_j~_NHNH2 (CH3)aN (Vl)
androsterone
0 + N-NH-C-CH=-N(CHz) 3 II
quaternaryammoniumderivative
(v)
(vt0
Derivatization can be used in conjunction with tandem mass spectrometry (MS/MS) to detect specific compound classes in complex mixtures. Collision-induced dissociation (CID) of molecular ions from phenols (e.g., VIII) is relatively inefficient, but the carbamate derivatives (e.g., IX) prepared with methyl isocyanate undergo ready dissociation with the characteristic neutral loss of methyl isocyanate (57 mass units) from the protonated molecular (X) ion formed under chemical ionization (CIMS) conditions. Thus an MS/MS neutral loss scan can be used to detect phenols in complex mixtures. 9
OH OCHaNCO (vB
H
÷
OCONHCHs +OCONHCHa O H = 0 CIMS [ ~ CID ~ ~ -I- CH3NCO 57 u (ix)
(x)
(xo
When mass spectrometry is interfaced to a separation technique (e.g., gas chromatography, high-performance liquid chromatography, capillary zone electrophoresis) derivatization may be used for enhancement of separability of the compounds of interest or to separate compounds of interest from other compounds present that interfere with the analysis. For example, if methyl ester derivatives are inadequately separated in a GC/MS analysis, other higher alkyl esters can be examined. Trimethylsilyl 8 G. C. DiDonato and K. L. Busch, Biomed. Mass Spectrom. 12, 364 (1985). 9 D. F. Hunt, J. Shabanowitz, T. M. Harvey, and M. L. Coates, J. Chromatogr. 271, 93 (1983).
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GENERALTECHNIQUES
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derivatives have been replaced with other alkylsilyl derivatives (e.g., isopropyldimethylsilyl) to facilitate the chromatographic separation in the simultaneous analysis of multiple eicosanoids by GC/MS. 1° Derivatization for modification o f the fragmentation behavior of molecules in the mass spectrometer is a powerful tool for structure elucidation. Such derivatization can be used to obtain molecular weight information or structural information. Compounds which fragment extensively and give low abundance molecular ions can be converted to derivatives which give more stable molecular ions by virtue of introduction of a chargestabilizing center. Alternatively, derivatives can be formed which fragment readily in a well-defined manner to give an abundant fragment ion characteristic of the molecular weight. An example of the latter is the tertbutyldimethylsilyl (TBDMS) derivative (e.g., XIII) which readily loses a tert-butyl radical from the molecular ion to give a very abundant (M - 57) + ion (XIV). This fragment ion retains the entire sample molecule structure and thus is indicative of the molecular weight.
R-OH
derivatization CH3 I CIHz MS + CHz I ~ R-O-Si~C-CH 3 ~- R-O=Si I
(xti)
I
I
CHa CHa
CHa
TBDMS derivative
(M-57) -I-
(xm)
(xlv)
Fragmentation modifying derivatives are also used extensively to enhance the formation of structurally informative ions. A classic problem is the identification of double-bond positions and branching points in longchain hydrocarbon groups. These chains fragment extensively with no position significantly favored for charge retention. A large number of derivatives have been examined for identification of double-bond positions, with two general approaches. One approach has been to carry out a chemical conversion on the double bond itself to introduce chargestabilizing, fragmentation directing groups. The second approach which is also useful for localization of branching and other substituents has been to introduce a charge-stabilizing group at the end of the chain. The pyridine ring appears to be the best group identified so far for this purpose. For fatty alcohols (XV) it is introduced as the nicotinic ester derivative (XVI) H and for fatty acids (XVII) as the picolinyl ester ( X V I I I ) . 12 These derivatives give a series of ions due to cleavage at each carbon position allowing l0 H. Miyazaki, M. Ishibashi, K. Yamashita, Y. Nishikawa, and M. Katori, Biomed. Mass Spectrom. 8, 521 (1981). ii W. Vetter and W. Meiser, Org. Mass Spectrom. 16, 118 (1981).
[ 15]
CHEMICAL DERIVATIZATION FOR MASS SPECTROMETRY
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identification of positions of unsaturation and branching as well as other features, such as, hydroxyl groups, additional carboxyl groups, and cyclopropane rings.
R-OH fatty alcohol (xv)
R-COOH fatty acid (xvn)
--- ~ C O O R nicotinicester (xvl)
=- ~ O C O R picolinylester (xviil)
A similar approach has been used to enhance the formation of sequence-specific ions in peptide sequencing using FAB ionization where a charge-stabilizing group (e.g., prolyl) ~3 or a preformed positive charge (via quaternization of the terminal amino group with methyl iodide)14 is introduced at the N terminus. These groups stabilize the positive charge on the N terminus of the peptide promoting the formation of a series of N-terminal sequence ions. Chemical derivatizations can also be used to determine the number and types of functional groups in a molecule. Examples from the peptide field include determining the number of carboxyl groups (and therefrom the number of acidic amino acid residues) by measuring the mass shift upon methyl ester formation, and determining the number of amino groups (and therefrom the number of lysine residues) by the mass shift following N-acetylation. Fragment ions containing the derivatized functional groups can be identified using the isotope doublet technique. For example, if acetylation is carried out using an equimolar mixture of acetic anhydride and hexadeuterated acetic anhydride, any ion containing an acetyl group will appear as a visually conspicuous equal-intensity doublet of peaks separated by three mass units. A similar approach can be used in conjunction with accurate mass measurements to yield an isotopic peak to corrobo-
12 D. J. Harvey, Biomed. Mass Spectrom. 9, 33 (1982). 13 D. F. Hunt, J. Shabanowitz, J. R. Yates, P. R. Griffin, andN. Z. Zhu, in "Mass Spectrometry of Biological Materials" (C. N. McEwen and B. S. Larsen, eds.), p. 169. Dekker, New York, 1990. 14 K. Biemann, this volume [25].
320
GENERAL TECHNIQUES
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rate an elemental composition determination. 15For example, a monoacetylated ion would have one peak whose elemental composition has only 1H and a corresponding peak 3 mass units higher with three 2H and three less ~H.
General Considerations in Chemical Derivatization A useful chemical derivatization reaction should be specific in that it modifies only the intended part of the molecule and should yield preferably a single stable product. It should be reasonably fast and simple to carry out and amenable to very small sample sizes. Ideally the derivatization reagents should also be reasonably stable and safe to handle. Few derivatization procedures meet all of these criteria. Chemical derivatization is a microscale form of synthetic organic chemistry where the objective is to achieve a 100% yield of a single product. While this goal is rarely achieved in macroscale synthesis, the use of highpurity, highly reactive reagents, often in large excess, makes it possible to approach this goal in many analytical derivatizations. The qualities of high purity and high reactivity of reagents present a difficult combination since highly reactive reagents are prone to decomposition and reaction with extraneous materials (e.g., atmospheric moisture). Great care is required to meet these criteria. Specially purified reagents packaged in small aliquots are preferred. Although reagents are less expensive in larger packages, such purchase is false economy if the reagents deteriorate before use. Use of large excesses of reagents to drive reactions to completion results in the need to remove the excess reagent. A common approach to this removal is evaporation resulting in concentration of any less volatile contaminants. Solvent evaporation also results in concentration of impurities. Therefore, solvents should be of the highest quality and volumes used kept to a minimum. The problems of concentration of low-volatility impurities in solvents and reagents are avoided with the use of vaporphase derivatization (see below).
Practical Aspects of Chemical Derivatization Most derivatization reactions are carded out in solution phase. The most widely used containers for such reactions are the tapered interior reaction vials.16 They are available in sizes ranging from 0.1 to 10.0 ml in 15K. Biemann, this volume [13]. 16Availablefrom a variety of sources, e.g.,React-VialsfromPierceChemicalCo., Rockford, IL; V-Vials from Wheaton, Millville, NJ.
[15]
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both clear and amber glass. The caps accommodate a variety of different types of liners, but the Teflon-faced rubber disks are most amenable to use with derivatization reactions. The caps are perforated to allow passage of a syringe needle through the liner without opening the container and exposing the contents to the atmosphere. Such perforation, however, destroys the integrity of the Teflon face and allows contact of the vial contents with the less chemically resistant bulk material of the cap liner. An alternative closure method is the use of all-Teflon valve closures. 17In the closed position, only Teflon is exposed to the vial interior. When the valve is opened, a rubber backup seal, which can be pierced with a syringe needle, seals out atmospheric moisture. To avoid the use of rubber, the backup seal (a cylindrical piece of silicone rubber) can be removed from its hole and a flow of inert gas purged through, which is used to protect the contents from the atmosphere. A variation of the conical reaction vial, which also facilitates the use of solvent extraction, is the Keele microreactor vial.18 The interior of this container contains a constriction which forms an "hourglass." Phase separation of microvolumes of liquid is facilitated by drawing the sample into the narrow constricted region using a syringe and needle. Separation of phases in microscale extractions can also be carried out in volumetric pipettor tips. Both phases are drawn into the pipettor allowing them to separate. The phases are then expelled sequentially into separate containers. The tapered reaction vials have smooth cylindrical sides and ground flat bottoms which allow close fit into bored aluminum heating blocks. Reaction mixtures can be stirred magnetically using miniature stirring paddles; heating block units are available commercially with built-in magnetic stirrers. 16The units can also be fitted with multiport evaporator units to evaporate samples under a stream of inert gas. An alternative method to evaporate samples to dryness employs the vacuum centrifuge device. ~9This device spins tubes in a centrifuge under vacuum to evaporate samples while continuously forcing the remaining solution to the bottom of the tube. This method has the advantage of concentrating the sample to a small area of the bottom of the tube rather than being dried over a larger surface area as occurs with evaporation under an inert gas stream. It also eliminates "bumping" during evaporation. 17 Mininert valves, Pierce Chemical Co., Rockford, IL. 18 A. B. Attygalle and E. D. Morgan, Anal. Chem. 58, 3054 (1986); available commercially from Wheaton, Millville, NJ. 19 Speedvac, Savant Instruments, Farmingdale, NY.
322
GENERAL TECHNIQUES
[15]
Tapered reaction vials have the disadvantage of being relatively expensive. For many applications, especially where large numbers of containers are used, autosampler vials with disposable glass inerts can be used. These vials are available in a wider range of sizes and styles (including smaller sizes) than can currently be found for tapered reaction vials. Another type of container, which is used for some of the less rigorous reactions in peptide analysis, is the polypropylene microcentrifuge tube. These are available either with snap tops or screw caps. 2° The latter type are available both with and without O-ring seals. For carrying out derivatizations, the screw caps which seal without O rings are preferable since the rubber O rings can yield contaminants. Polypropylene tubes are preferable to glass for those compounds which are prone to loss by adsorption on glass surfaces (e.g., basic peptides). Alternatively, the glass surfaces can be silanized to mask the adsorptive sites with methylated silyl groups. The most convenient way to silanize glassware is to use the vacuum oven technique where the glassware is heated in the presence ofhexamethyldisilazane vapor. 21The solution silanization procedure can be used for small quantities of glassware which do not warrant the dedication of a vacuum oven apparatus (some workers feel that the Solution method is better regardless). In the solution method, the thoroughly cleaned and oven-dried glassware is treated for 30 min with a 5% solution of dimethyldichlorosilane in toluene. It is then washed sequentially with toluene and anhydrous methanol. After air-drying under a fume hood, the glassware is oven-dried before use. Some containers, such as autosampler vials and polypropylene tubes, are disposable. More expensive containers such as the tapered reaction vials must be cleaned and reused. For high-sensitivity analysis, conventional cleaning is often followed by baking to pyrolyze any remaining organic material. Laboratories processing large numbers of containers have found it convenient to use a standard domestic self-cleaning oven for this purpose. Pyrolytic cleaning destroys surface silanization thus necessitating resilanization prior to use. Reagent measurement and delivery of less reactive reagents and those not sensitive to atmospheric exposure can be performed with any of the many types of micropipettes. More reactive reagents are easily transferred using microliter syringes. The "gas-tight" type with Teflon-tipped plungers are easy to use and do not suffer from reagent soaking into the space around the metal plunger. If all exposure to metal must be avoided, the 20 Sarstedt, Newton, NC; Bio-Rad, Richmond, CA. 21 D. C. Fenimore, C. M. Davis, J. H. Whitford, and C. A. Harrington, Anal. Chem. 48, 2289 0976).
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CHEMICAL DERIVATIZATION FOR MASS SPECTROMETRY
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removable needle type can be used with a disposable glass micropipette substituted for the needle. An alternative measuring device where the reagent contacts only glass is a modified Eppendorf-type pipettor where the disposable polypropylene tip has been cut off and a melting point capillary whose tip has been drawn out in a flame ~8 is forced into the opening. When this device is used with corrosive reagents, the metal piston inside the pipettor must be cleaned after each use to prevent its deterioration.
Derivatization with Vapor-Phase Reagents An alternative to derivatization in solution phase is to use vapor-phase reagents acting upon a sample film dried on a surface. This approach, used to acylate steroid samples on platinum gauze 20 years ago, 22 has been reintroduced by Biemann's laboratory. 23 The use of vapor-phase reagents acting upon a dried sample allows multistep reactions to be carried out without evaporation of reagents and solvents (and attendant accumulation of impurities) and without sample transfers (and attendant losses). In the recent embodiment of the technique, 24,25 a few microliters of the sample solution are placed in a melting point capillary which contains a small indentation to hold the liquid in place. Evaporation of the solvent under vacuum deposits the sample on a small area of the inner surface of the capillary. The sample-containing capillaries are then placed in a reaction vessel 25 made from a commercially available vacuum hydrolysis tube. 26 The vacuum hydrolysis tube is used in a horizontal position with indentations added to support the capillaries lying horizontally (so that their ends do not contact the tube surface) and with the addition of a reagent well. The reagent (ca. 200 ~1) is added to the well and the sample-containing tubes inserted into the vessel. After cooling the reagent, the vessel is evacuated, sealed, and then placed in an oven at the appropriate temperature. After the reaction period, the reagent in the well is cooled again and the excess vapor removed by evaporation. Multiple derivatizations can be carried out by placing the sample capillaries in successive vessels containing different reagents. Following derivatization the sample tubes can be directly transferred to a Caplan and Cronin27-type gas chromatograph injector for GC/MS analysis or dissolved in a small volume of solvent for probe analysis. Transfers of very small volumes of solution from these 22 E. C. Homing and B. F. Maume, J. Chromatatogr. Sci. 7, 411 (1969). 23 j. E. Vath, M. Zollinger, and K. Biemann, Fresenius Z. Anal. Chem. 331, 248 (1988). 24 K. Biemann, this volume [18]. 25 C. E. Costello and J. E. Vath, this volume [40]. 26 Pierce Chemical Co., Rockford, IL. 27 p. j. Caplan and D. A. Cronin, J. Chromatogr. 267, 19 (1983).
324
GENERAL TECHNIQUES
[15]
tubes can be carried out with a microliter syringe with a fused-silica capillary "needle," such as is used for on-column injections in capillary gas chromatogrpahy. Derivatization reactions which have been carried out by this method include trifluoroacetylation, acetylation, esterification (of a peptide via an intermediate azalactone formed with acetic anhydride), quaternization of amino groups, borane reduction of peptides and gangliosides, and trimethylsilylation. 23 A variety of other reactions should also be amenable to this approach.
On-Probe Derivatization in FAB Analysis For mass spectrometric analysis by FAB techniques, derivatization reactions may also be carried out directly on the sample in the liquid matrix on the probe. Examples of such reactions include acetylation, esterification, disulfide reduction, and oxidations where the reagents are added directly to the sample matrix. In some cases, the matrix itself can serve as a derivatization reagent, i.e., the reduction of disulfide bonds in a thioglycerol matrix. Using on-probe derivatization, it is possible to obtain spectra of a compound and one or more derivatives from the same sample loading on the probe. Desirable properties in a derivatization reagent for this purpose are rapid reaction at ambient temperature, relative insensitivity to atmospheric oxygen and moisture, and volatility for removal of excess reagent. Excess reagents can be removed by evaporation in the vacuum lock during probe insertion or, to protect the mass spectrometer from excessive exposure to reagents, in a separately pumped vacuum system.
Derivatization Methods The following is a sampling of general-purpose derivatization reaction procedures. These methods have been generalized from a collection of methods optimized for specific sample types, 3 from recent literature, and from collected "recipes" whose original sources are difficult to determine. In most cases, exact quantities are not stated. The reagents are generally used in large excess but on an analytical scale (e.g., nano- to picomole) even microliter quantities of reagents usually represent a large excess. The user should, however, check that the reagent amount used is in excess of the amount of sample being derivatized, Different compound types will require specifically optimized conditions for optimum derivative yields, especially for high-sensitivity quantitation; for these specific conditions
[15]
CHEMICAL DERIVATIZATION FOR MASS SPECTROMETRY
325
the reader is referred to later chapters in this volume and to the previously referenced sources. 2'3 The methods given here should be useful for qualitative work and as a starting point to develop optimized methods.
Methyl Ester Formation A solution of 2 N methanolic HCI is prepared by adding dropwise 150/.d of acetyl chloride to 1 ml of methanol. The acetyl chloride is conveniently measured using a disposable glass micropipette and added to the methanol in a glass test tube with mixing on a Vortex mixer. The solution is allowed to stand at room temperature for 5-10 rain and then added to the dried sample. After standing 1-2 hr at room temperature (a much shorter time may be adequate), the excess reagent is removed by evaporation under an inert gas stream or under vacuum. The esterification reagent should be freshly prepared; old solutions may contain chloromethane which could cause extraneous reactions. Methyl esters can also be prepared using diazomethane. This method avoids the risk of methanolysis of susceptible functional groups (e.g., amide groups in peptides). Diazomethane is highly toxic and explosive but can be handled safely in small quantities in a hood. Small quantities (less than 1 mmol) for derivatization can be prepared using a generator as described by Fales et ai.28(now commercially available) 26,29'3°which avoids the inconvenience and hazards of distillation. The diazomethane is produced by adding alkali via a syringe port in the generator to an aqueous solution of N-methyl-N-nitroso-N'-nitroguanidine (MNNG) contained in the inner of two concentric reservoirs (the more common Diazald precursor should not be used because it requires heating). The generated diazomethane dissolves in diethyl ether contained in the outer reservoir. Diazoethane can be prepared in the same manner using the homologous precursor. Deuterodiazomethane (which yields [2H2]methyl derivatives) can be prepared from MNNG by substituting 2H-labeled (-O2H) 2-(2-ethoxyethoxy)ethanol for the water and sodium deuteroxide as alkali. The derivatization is carried out by dissolving the sample in the ether solution of diazomethane. If necessary, methanol can be used to dissolve the sample. The reaction with carboxyl groups is very rapid and the persistence of the yellow color of diazomethane indicates excess reagent. For larger sample sizes, the by-product nitrogen can be observed as bubbles. The excess reagent and solvent are removed by evaporation (in a 28 H. M. Fales, T. M. Jaouni, and J. F. Babashak, Anal. Chem. 45, 2302 (1973). 29 Wheaton, Millville, NJ. 30 Aldrich Chemical Co., Milwaukee, WI.
326
GENERAL TECHNIQUES
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hood). Diazomethane can also react with carbonyl groups and double bonds to give extraneous products.
Acetylation Acetylations of OH (both alcohol and phenol) and NH (amine) groups can be carried out using acetic anhydride with either base or acid catalysis. Common reagents include 1 : 1 (v/v) acetic anhydride/pyridine or acetic anhydride/acetic acid. Dimethylaminopyridine, a much stronger catalyst than pyridine, can be used in much smaller amounts (e.g., 2% in acetic anhydride). Reactions are usually complete in 30-60 min (often less) at room temperature or with only mild heating. Peptide amino groups can be acetylated in a few minutes at room temperature with an equal volume mixture of acetic anhydride and methanol. Acetylated peaks can be identified as M,M + 3 doublets by use of 1:1 acetic anhydride/[2H6]acetic anhydride.
Trifluoroacetylation Trifluoroacetyl derivatives are commonly prepared from OH and NH groups; the amide derivatives are, in general, more stable than the ester derivatives. Trifluoracetic anhydride. (TFAA), trifluoroacetylimidazole (TFAI), or N-methylbis(trifluoroacetamide) (MBTFA) can be used as reagents. TFAA is used with either acidic (typically TFA) or basic (typically trimethylamine) catalysis. Both TFAA and TFAI methods yield nonvolatile by-products which normally require isolation of the derivative by solvent extraction. The most convenient general-purpose reagent is MBTFA. The sample is dissolved in the reagent or in a mixture (1 : 1, v/v) of the reagent and pyridine and heated at 60° for 1 hr or more. For GC/MS analysis, the mixture can be injected directly since the by-product is the innocuous and volatile N-methyltrifluoroacetamide. Alternatively, the excess reagent and by-product can be evaporated under vacuum to isolate the derivative (as long as the derivative is less volatile than the byproduct). Methyl trifluoroacetate can be used to trifluoroacetylate amino groups (e.g., in peptide methyl esters) under mild conditions) ~ The sample is dissolved in an equal volume mixture of methanol and methyl trifluoroacetate. Triethylamine is added to adjust the pH to above 8 (checked with moistened pH paper) and the solution allowed to stand at room temperature overnight. The excess reagents can be removed by evaporation. 31 S. A. Carr, W. C. Herlihy, and K. Biemann, Biomed. Mass Spectrom. 8, 51 (1981).
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CHEMICAL DERIVATIZATION FOR MASS SPECTROMETRY
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Trimethylsilylation A variety of reagents are available for trimethylsilylation. A good general-purpose reagent which will silylate alcohols, phenols, amines, carboxylic acids, and thiols is N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA). The by-products trimethylsilyltrifluoroacetamide and trifluoroacetamide are quite volatile, as is the reagent itself. The sample to be silylated is dissolved in the reagent or, if necessary, in silylation-grade acetonitrile, dimethylformamide, or pyridine followed by addition of excess reagent. The reaction often proceeds well at room temperature but may require heating. A more powerful reagent contains 1% trimethylchlorosilane (TMCS) in BSTFA. For GC/MS analysis, the derivatization mixture is normally injected directly.
tert-Butyldimethylsilylation The most convenient reagent for forming tert-butyldimethylsilyl (TBDMS) derivatives is N-methyl-N-(tert-butyldimethylsilyl)trifluoroacetamide (MTBSTFA). Its use is similar to BSTFA but often requires more vigorous reaction conditions to transfer the bulkier silyl group. MTBSTFA containing 1% of the corresponding chlorosilane (available commercially)32is probably the best first choice for most compounds. The TBDMS group is much more stable toward hydrolysis than the TMS group; unlike TMS derivatives, the TBDMS derivatives can be isolated, if desired, for direct probe analysis.
Methoxime Formation Methoxime derivatives are used to reduce the polarity and hydrogen bond acceptor potential of aldehyde and ketone carbonyl groups. A disadvantage of the derivative is the possible formation of chromatographically separable syn and anti products due to restricted rotation about the methoxime double bond. The derivative is prepared by adding a 2% solution of methoxyamine hydrochloride in pyridine to the sample. Reaction conditions vary for different compounds but are typically several hours to overnight at room temperature or one to a few hours at 60°. The excess reagent is not volatile, therefore the derivatized sample is usually recovered by adding water (or aqueous NaCI) and extracting with ethyl acetate. In a multistep derivatization involving water-sensitive derivatives, the methoximation must be performed first. 32 Regis Chemical Company, Morton Grove, IL.
328
GENERAL TECHNIQUES
[15]
On-Probe Derivatization Methods On-probe derivatization methods are commonly used in FAB analysis, but can be carried out on sample in the probe capillary in EI and CI probe analysis. Thus, reaction conditions are less stringent than for conventional derivatizations. The following listed below are commonly used on-probe procedures. Methyl Ester Formation. One microliter of 2N methanolic HC1 is added to the sample (or in the case of FAB, the sample/matrix mixture) on the probe tip and allowed to stand for a few minutes at room temperature. The sample is then reinserted into the vacuum lock and the excess reagents pumped away. If the ester peaks are not observed, the derivatization can be repeated for a longer period. Acetylation of Amino Groups in Peptides. A 0.5-/zl aliquot of 1:1 (v/v) methanol/acetic anhydride and 0.5/zl of pyridine are added to the sample on the probe tip and allowed to stand at room temperature for a few minutes. The excess reagents are then removed in the vacuum lock prior to analysis. Reduction of Disulfide Bonds in Peptides. Raising the pH of a sample in thioglycerol or dithiothreitol (DTT)/dithioerythritol (DTE) matrix by addition of 0.5/zl 28% aqueous ammonia will promote reduction by the thiol matrix. Alternatively, 1/zl of either 1 : 1 (v/v) 0.2 M N-ethylmorpholine buffer (pH 8.5)/I M DTT or 1:1 (v/v) aqueous ammonia (28%)/ thioglycerol is added and allowed to stand for a few minutes at room temperature. 33 The sample is then acidified with HCI or trifluoroacetic acid, and FAB matrix added if necessary, prior to removal of excess reagent in the vacuum lock and analysis. Performic Acid Oxidation. Pefformic acid is prepared by adding 5% (by volume) of 30% hydrogen peroxide to 99% formic acid and allowing the solution to stand at room temperature for 2 hr. One microliter of the solution is added to the sample in glycerol matrix and allowed to stand for several minutes prior to analysis. Summary and Future Prospects Chemical derivatization in relation to mass spectrometry has evolved from primarily volatility considerations to a primary emphasis upon manipulation of the ion chemistry in the mass spectrometer to increase the information yield. The advent of FAB methods at first appeared to obviate the need for derivatization, but now it is recognized that FAB presents 33 H. Rodriqucz, B. Nevins, and J. Chakcl, in "Techniques in Protein Chemistry" (T. E. Hugli, ed.), p. 186. Academic Press, San Diego, 1989.
[16]
DEUTERIUM EXCHANGE PROCEDURES
329
new opportunities to exploit derivatization. Likewise, the rapidly developing field of tandem mass spectrometry creates additional opportunities for the creative use of chemistry in conjunction with mass spectrometry to increase the ability to solve structural problems. In the area of protein structure, for example, mass spectrometry has already established itself as the "third leg of the stool" of protein primary structure methods (along with Edman and DNA sequencing). To date, however, mass spectrometry has been little exploited for other than determination of primary covalent structure. In conjuction with creative uses of chemical modifications (derivatizations), mass spectrometry offers the potential to yield three-dimensional information as well. Chemical linkage experiments can yield distance geometry measurements which, in conjunction with computer molecular graphics, can be used to refine three-dimensional structures. As a further extension, chemical trapping of intermediate states offers the potential to gain information on molecular dynamics. Thus, there appears to be a wide range of future applications for chemical derivatization in the broadest sense of the term. Just as with protein chemistry, whose demise was prematurely announced some years ago, 34 chemical derivatization in relation to mass spectrometry is far from dead. 34 A. D. B. Malcolm,
Nature (London) 275,
90 (1978).
[16] I n t r o d u c t i o n o f D e u t e r i u m b y E x c h a n g e for Measurement by Mass Spectrometry By
JAMES A. MCCLOSKEY
The chemical incorporation of deuterium followed by measurement of the intact product by mass spectrometry has for a number of years played a major role in three, often overlapping areas: the structural characterization of molecules of unknown structure; to gain information on the mechanisms of chemical or biological reactions; and as an aid in the interpretation of mass spectra. 1 In each of these areas mass spectrometry offers several general advantages: (1) relatively routine measurements can be made in the sample quantity range 10-11-10-6 g, sometimes in mixtures or without extensive sample purification; (2) the sites of labeling can often be deterI H. Budzikiewicz, C. Djerassi, and D. H. Williams, " S t r u c t u r e Elucidation of Natural Products by M a s s S p e c t r o m e t r y , " Vol. 1, Alkaloids, Chap. 2. Holden-Day, San Francisco, 1964.
METHODS IN ENZYMOLOGY, VOL. 193
Copyright © 1990by AcademicPress, Inc. All fights of reproduction in any form reserved.
GENERAL TECHNIQUES
[15]
We have found that these salt mixtures age and their FAB spectra will change over time. It is, therefore, necessary to adjust the salt concentrations and the reference table when a new calibration mixture is made. Conclusions There are wide varieties of reference compounds available for use in mass spectrometry and it is impossible in this brief space to give all the details of their use. Table I summarizes the commonly used reference compounds and Appendix 2 at the end of the volume includes tables with accurate masses of reference compound peaks.
[15] C h e m i c a l D e r i v a t i z a t i o n for M a s s S p e c t r o m e t r y
By DANIEL R. KNAPP Introduction Chemical derivatization has played an imporant role in organic mass spectrometry from the earliest practice of the technique.1 Although the major impetus for its early use was to confer the necessary volatility to sample compounds, chemical derivatization was also used to increase the information yield from mass spectral data. With decades of progress in mass spectrometry and the development of a wide range of new techniques, chemical derivatization still plays an important role. This chapter will give an overview of the use of chemical derivatization in mass spectrometry with particular emphasis on analytical strategy aspects. General considerations with respect to the practical aspects of chemical derivatizations and some general methods will also be described. More specific applications are given in later chapters in this volume, as well as in a recent review 2 and monograph. 3 i K. Biemann, "Mass Spectrometry, Organic Chemical Applications." McGraw-Hill, New York, 1962. 2 R. J. Anderegg, Mass Spectrom. Rev. 7, 395 (1988). 3 D. R. Knapp, "Handbook of Analytical Derivatization Reactions." Wiley, New York, 1979.
METHODS IN ENZYMOLOGY, VOL. 193
Copyright © 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
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Reasons for Use of Chemical Derivatization in Mass Spectrometry The reasons for utilizing chemical derivatization in relation to mass spectrometry can be summarized as follows: (1) enhancement of volatility; (2) degradation of the sample molecule to smaller subunits; (3) enhancement of detectability; (4) enhancement of separability; (5) modification of fragmentation: (a) enhancement of molecular weight related ions and (b) enhancement of structurally informative ions; (6) determination of functional groups. Volatility enhancement was a requirement for many samples in the early days of mass spectrometry when the available inlet systems required significant vapor pressure. In current practice, derivatization for volatility enhancement is used primarily in relation to interfaced gas chromatography-mass spectrometry (GC/MS) where such derivatization serves to enable or improve the gas chromatography. Vapor pressure of a compound is influenced by intermolecular attractions due to dispersion (van der Waals) forces, ionic interactions, and hydrogen bonds. The total dispersion forces increase with molecular size and little can be done with respect to these forces to increase volatility other than reduce the size of the molecule (see below). Chemical derivatization for volatility enhancement is aimed at reducing ionic and hydrogen bond interactions by conversion of ionizable groups to nonionizable derivatives (e.g., carboxyl groups to esters); replacing hydrogens bound to heteroatoms (N-H, O-H, S-H) with alkyl, acyl, silyl, or other groups; and reducing the polarity of hydrogen bond accepting groups (e.g., conversion of carbonyls to methoximes). An example of a multistep derivatization to increase volatility is the conversion of peptides (1) to the volatile polyamino alcohol trimethylsilyl ethers (11).4 Not only are these derivatives sufficiently volatile for GC/ MS, but they also have the added advantage of yielding stable, structurally informative ammonium-type ions in the mass spectrum. Thus, these derivatives (as is often the objective) serve a dual purpose. R O I II H=N-(CH-C)n--OH
peptide
1. acylation (R'CO-) R 2. reduction I ~ R'CH=NH-(CH-CH2)n--OTMS 3. trimethylsilylation
(I)
trimethylsilyl (TMS) polyamino alcohol
(ii)
Degradation of sample molecules by chemical means was originally employed for volatility consideration, i.e., large molecules were degraded to smaller constituent molecules with sufficient vapor pressure to be ana4 K. B i e m a n n , this v o l u m e [18].
316
GENERAL TECHNIQUES
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lyzed. In current practice, degradations are still used to identify constituent subunits, but molecular size reductions are driven more by instrument mass range limits than volatility. In the case of tandem mass spectrometry, the need for degradation to smaller size fragments may be motivated by molecular size limits of current ion dissociation technology rather than the analyzer mass range. An example of a contemporary use of degradation is the acetolysis of glycoproteins to cleave off the oligosaccharide components as peracetylated carbohydratesS: Glycoprotein
acetolysis
~Peracetylatedoligosaccharides
Derivatization for detectability enhancement involves chemical conversions to promote the formation of stable charged species with either little fragmentation or characteristic fragmentation behavior to yield intense well-defined ions. An example is the attachment of an electrophoric moiety to promote the formation of negative ions i n " negative ion chemical ionization" (NICI) mass spectrometry. This approach has been used, in particular, for eicosanoids for high sensitivity quantitation. 6 For example, prostaglandin E 2 ( m ) is converted in three steps to the methoxime (MO), trimethylsilyl (TMS), pentafluorobenzyl (PFB) derivative (IV) which exhibits good gas chromatographic behavior. Under NICI conditions the derivative captures an electron to yield a negatively charged molecular radical anion which readily loses a pentafluorobenzyl radical to form a relatively stable carboxylate anion. The resulting mass spectrum exhibits an intense ( M - 181)- peak which can be monitored by GC/MS selectedion monitoring for very sensitive, and selective, detection and quantitation of eicosanoids in biological samples. 0 ~ ~ C O O H
1. methoximation 2. pentafluorobenzyl esterification ~ 3. silylation
HO
OH Prostaglandin E2
(no
N-OCH~ \\ ~ " ~ , ,' TMSO
~COOCH=CaFs J
. i OTM$
MO-PFB-TMS Derivative
(iv)
The advent of fast atom bombardment (FAB) ionization 7 has led to the use of chemical derivatization for detectability enhancement by the 5 A. Dell, this volume [35]. 6 I. Blair, this series, Vol. 187, p. 13. 7 In this chapter, the term, FAB, is used generically to include ion bombardment of samples in liquid matrices.
[15]
CHEMICAL DERIVATIZATION FOR MASS SPECTROMETRY
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intentional introduction of a charged group into the molecule, which is just the opposite of the traditional masking of polar groups. For example, the ketosteroid androsterone (V), which is undetectable under normal FAB conditions (glycerol, 4/zg//zl) in the native state, gives an intense molecular ion as the quaternary ammonium derivative (VII) resulting from derivatization with Girard's reagent T (VI). s
0 H
O +_j~_NHNH2 (CH3)aN (Vl)
androsterone
0 + N-NH-C-CH=-N(CHz) 3 II
quaternaryammoniumderivative
(v)
(vt0
Derivatization can be used in conjunction with tandem mass spectrometry (MS/MS) to detect specific compound classes in complex mixtures. Collision-induced dissociation (CID) of molecular ions from phenols (e.g., VIII) is relatively inefficient, but the carbamate derivatives (e.g., IX) prepared with methyl isocyanate undergo ready dissociation with the characteristic neutral loss of methyl isocyanate (57 mass units) from the protonated molecular (X) ion formed under chemical ionization (CIMS) conditions. Thus an MS/MS neutral loss scan can be used to detect phenols in complex mixtures. 9
OH OCHaNCO (vB
H
÷
OCONHCHs +OCONHCHa O H = 0 CIMS [ ~ CID ~ ~ -I- CH3NCO 57 u (ix)
(x)
(xo
When mass spectrometry is interfaced to a separation technique (e.g., gas chromatography, high-performance liquid chromatography, capillary zone electrophoresis) derivatization may be used for enhancement of separability of the compounds of interest or to separate compounds of interest from other compounds present that interfere with the analysis. For example, if methyl ester derivatives are inadequately separated in a GC/MS analysis, other higher alkyl esters can be examined. Trimethylsilyl 8 G. C. DiDonato and K. L. Busch, Biomed. Mass Spectrom. 12, 364 (1985). 9 D. F. Hunt, J. Shabanowitz, T. M. Harvey, and M. L. Coates, J. Chromatogr. 271, 93 (1983).
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GENERALTECHNIQUES
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derivatives have been replaced with other alkylsilyl derivatives (e.g., isopropyldimethylsilyl) to facilitate the chromatographic separation in the simultaneous analysis of multiple eicosanoids by GC/MS. 1° Derivatization for modification o f the fragmentation behavior of molecules in the mass spectrometer is a powerful tool for structure elucidation. Such derivatization can be used to obtain molecular weight information or structural information. Compounds which fragment extensively and give low abundance molecular ions can be converted to derivatives which give more stable molecular ions by virtue of introduction of a chargestabilizing center. Alternatively, derivatives can be formed which fragment readily in a well-defined manner to give an abundant fragment ion characteristic of the molecular weight. An example of the latter is the tertbutyldimethylsilyl (TBDMS) derivative (e.g., XIII) which readily loses a tert-butyl radical from the molecular ion to give a very abundant (M - 57) + ion (XIV). This fragment ion retains the entire sample molecule structure and thus is indicative of the molecular weight.
R-OH
derivatization CH3 I CIHz MS + CHz I ~ R-O-Si~C-CH 3 ~- R-O=Si I
(xti)
I
I
CHa CHa
CHa
TBDMS derivative
(M-57) -I-
(xm)
(xlv)
Fragmentation modifying derivatives are also used extensively to enhance the formation of structurally informative ions. A classic problem is the identification of double-bond positions and branching points in longchain hydrocarbon groups. These chains fragment extensively with no position significantly favored for charge retention. A large number of derivatives have been examined for identification of double-bond positions, with two general approaches. One approach has been to carry out a chemical conversion on the double bond itself to introduce chargestabilizing, fragmentation directing groups. The second approach which is also useful for localization of branching and other substituents has been to introduce a charge-stabilizing group at the end of the chain. The pyridine ring appears to be the best group identified so far for this purpose. For fatty alcohols (XV) it is introduced as the nicotinic ester derivative (XVI) H and for fatty acids (XVII) as the picolinyl ester ( X V I I I ) . 12 These derivatives give a series of ions due to cleavage at each carbon position allowing l0 H. Miyazaki, M. Ishibashi, K. Yamashita, Y. Nishikawa, and M. Katori, Biomed. Mass Spectrom. 8, 521 (1981). ii W. Vetter and W. Meiser, Org. Mass Spectrom. 16, 118 (1981).
[ 15]
CHEMICAL DERIVATIZATION FOR MASS SPECTROMETRY
319
identification of positions of unsaturation and branching as well as other features, such as, hydroxyl groups, additional carboxyl groups, and cyclopropane rings.
R-OH fatty alcohol (xv)
R-COOH fatty acid (xvn)
--- ~ C O O R nicotinicester (xvl)
=- ~ O C O R picolinylester (xviil)
A similar approach has been used to enhance the formation of sequence-specific ions in peptide sequencing using FAB ionization where a charge-stabilizing group (e.g., prolyl) ~3 or a preformed positive charge (via quaternization of the terminal amino group with methyl iodide)14 is introduced at the N terminus. These groups stabilize the positive charge on the N terminus of the peptide promoting the formation of a series of N-terminal sequence ions. Chemical derivatizations can also be used to determine the number and types of functional groups in a molecule. Examples from the peptide field include determining the number of carboxyl groups (and therefrom the number of acidic amino acid residues) by measuring the mass shift upon methyl ester formation, and determining the number of amino groups (and therefrom the number of lysine residues) by the mass shift following N-acetylation. Fragment ions containing the derivatized functional groups can be identified using the isotope doublet technique. For example, if acetylation is carried out using an equimolar mixture of acetic anhydride and hexadeuterated acetic anhydride, any ion containing an acetyl group will appear as a visually conspicuous equal-intensity doublet of peaks separated by three mass units. A similar approach can be used in conjunction with accurate mass measurements to yield an isotopic peak to corrobo-
12 D. J. Harvey, Biomed. Mass Spectrom. 9, 33 (1982). 13 D. F. Hunt, J. Shabanowitz, J. R. Yates, P. R. Griffin, andN. Z. Zhu, in "Mass Spectrometry of Biological Materials" (C. N. McEwen and B. S. Larsen, eds.), p. 169. Dekker, New York, 1990. 14 K. Biemann, this volume [25].
320
GENERAL TECHNIQUES
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rate an elemental composition determination. 15For example, a monoacetylated ion would have one peak whose elemental composition has only 1H and a corresponding peak 3 mass units higher with three 2H and three less ~H.
General Considerations in Chemical Derivatization A useful chemical derivatization reaction should be specific in that it modifies only the intended part of the molecule and should yield preferably a single stable product. It should be reasonably fast and simple to carry out and amenable to very small sample sizes. Ideally the derivatization reagents should also be reasonably stable and safe to handle. Few derivatization procedures meet all of these criteria. Chemical derivatization is a microscale form of synthetic organic chemistry where the objective is to achieve a 100% yield of a single product. While this goal is rarely achieved in macroscale synthesis, the use of highpurity, highly reactive reagents, often in large excess, makes it possible to approach this goal in many analytical derivatizations. The qualities of high purity and high reactivity of reagents present a difficult combination since highly reactive reagents are prone to decomposition and reaction with extraneous materials (e.g., atmospheric moisture). Great care is required to meet these criteria. Specially purified reagents packaged in small aliquots are preferred. Although reagents are less expensive in larger packages, such purchase is false economy if the reagents deteriorate before use. Use of large excesses of reagents to drive reactions to completion results in the need to remove the excess reagent. A common approach to this removal is evaporation resulting in concentration of any less volatile contaminants. Solvent evaporation also results in concentration of impurities. Therefore, solvents should be of the highest quality and volumes used kept to a minimum. The problems of concentration of low-volatility impurities in solvents and reagents are avoided with the use of vaporphase derivatization (see below).
Practical Aspects of Chemical Derivatization Most derivatization reactions are carded out in solution phase. The most widely used containers for such reactions are the tapered interior reaction vials.16 They are available in sizes ranging from 0.1 to 10.0 ml in 15K. Biemann, this volume [13]. 16Availablefrom a variety of sources, e.g.,React-VialsfromPierceChemicalCo., Rockford, IL; V-Vials from Wheaton, Millville, NJ.
[15]
CHEMICAL DERIVATIZATION FOR MASS SPECTROMETRY
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both clear and amber glass. The caps accommodate a variety of different types of liners, but the Teflon-faced rubber disks are most amenable to use with derivatization reactions. The caps are perforated to allow passage of a syringe needle through the liner without opening the container and exposing the contents to the atmosphere. Such perforation, however, destroys the integrity of the Teflon face and allows contact of the vial contents with the less chemically resistant bulk material of the cap liner. An alternative closure method is the use of all-Teflon valve closures. 17In the closed position, only Teflon is exposed to the vial interior. When the valve is opened, a rubber backup seal, which can be pierced with a syringe needle, seals out atmospheric moisture. To avoid the use of rubber, the backup seal (a cylindrical piece of silicone rubber) can be removed from its hole and a flow of inert gas purged through, which is used to protect the contents from the atmosphere. A variation of the conical reaction vial, which also facilitates the use of solvent extraction, is the Keele microreactor vial.18 The interior of this container contains a constriction which forms an "hourglass." Phase separation of microvolumes of liquid is facilitated by drawing the sample into the narrow constricted region using a syringe and needle. Separation of phases in microscale extractions can also be carried out in volumetric pipettor tips. Both phases are drawn into the pipettor allowing them to separate. The phases are then expelled sequentially into separate containers. The tapered reaction vials have smooth cylindrical sides and ground flat bottoms which allow close fit into bored aluminum heating blocks. Reaction mixtures can be stirred magnetically using miniature stirring paddles; heating block units are available commercially with built-in magnetic stirrers. 16The units can also be fitted with multiport evaporator units to evaporate samples under a stream of inert gas. An alternative method to evaporate samples to dryness employs the vacuum centrifuge device. ~9This device spins tubes in a centrifuge under vacuum to evaporate samples while continuously forcing the remaining solution to the bottom of the tube. This method has the advantage of concentrating the sample to a small area of the bottom of the tube rather than being dried over a larger surface area as occurs with evaporation under an inert gas stream. It also eliminates "bumping" during evaporation. 17 Mininert valves, Pierce Chemical Co., Rockford, IL. 18 A. B. Attygalle and E. D. Morgan, Anal. Chem. 58, 3054 (1986); available commercially from Wheaton, Millville, NJ. 19 Speedvac, Savant Instruments, Farmingdale, NY.
322
GENERAL TECHNIQUES
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Tapered reaction vials have the disadvantage of being relatively expensive. For many applications, especially where large numbers of containers are used, autosampler vials with disposable glass inerts can be used. These vials are available in a wider range of sizes and styles (including smaller sizes) than can currently be found for tapered reaction vials. Another type of container, which is used for some of the less rigorous reactions in peptide analysis, is the polypropylene microcentrifuge tube. These are available either with snap tops or screw caps. 2° The latter type are available both with and without O-ring seals. For carrying out derivatizations, the screw caps which seal without O rings are preferable since the rubber O rings can yield contaminants. Polypropylene tubes are preferable to glass for those compounds which are prone to loss by adsorption on glass surfaces (e.g., basic peptides). Alternatively, the glass surfaces can be silanized to mask the adsorptive sites with methylated silyl groups. The most convenient way to silanize glassware is to use the vacuum oven technique where the glassware is heated in the presence ofhexamethyldisilazane vapor. 21The solution silanization procedure can be used for small quantities of glassware which do not warrant the dedication of a vacuum oven apparatus (some workers feel that the Solution method is better regardless). In the solution method, the thoroughly cleaned and oven-dried glassware is treated for 30 min with a 5% solution of dimethyldichlorosilane in toluene. It is then washed sequentially with toluene and anhydrous methanol. After air-drying under a fume hood, the glassware is oven-dried before use. Some containers, such as autosampler vials and polypropylene tubes, are disposable. More expensive containers such as the tapered reaction vials must be cleaned and reused. For high-sensitivity analysis, conventional cleaning is often followed by baking to pyrolyze any remaining organic material. Laboratories processing large numbers of containers have found it convenient to use a standard domestic self-cleaning oven for this purpose. Pyrolytic cleaning destroys surface silanization thus necessitating resilanization prior to use. Reagent measurement and delivery of less reactive reagents and those not sensitive to atmospheric exposure can be performed with any of the many types of micropipettes. More reactive reagents are easily transferred using microliter syringes. The "gas-tight" type with Teflon-tipped plungers are easy to use and do not suffer from reagent soaking into the space around the metal plunger. If all exposure to metal must be avoided, the 20 Sarstedt, Newton, NC; Bio-Rad, Richmond, CA. 21 D. C. Fenimore, C. M. Davis, J. H. Whitford, and C. A. Harrington, Anal. Chem. 48, 2289 0976).
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CHEMICAL DERIVATIZATION FOR MASS SPECTROMETRY
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removable needle type can be used with a disposable glass micropipette substituted for the needle. An alternative measuring device where the reagent contacts only glass is a modified Eppendorf-type pipettor where the disposable polypropylene tip has been cut off and a melting point capillary whose tip has been drawn out in a flame ~8 is forced into the opening. When this device is used with corrosive reagents, the metal piston inside the pipettor must be cleaned after each use to prevent its deterioration.
Derivatization with Vapor-Phase Reagents An alternative to derivatization in solution phase is to use vapor-phase reagents acting upon a sample film dried on a surface. This approach, used to acylate steroid samples on platinum gauze 20 years ago, 22 has been reintroduced by Biemann's laboratory. 23 The use of vapor-phase reagents acting upon a dried sample allows multistep reactions to be carried out without evaporation of reagents and solvents (and attendant accumulation of impurities) and without sample transfers (and attendant losses). In the recent embodiment of the technique, 24,25 a few microliters of the sample solution are placed in a melting point capillary which contains a small indentation to hold the liquid in place. Evaporation of the solvent under vacuum deposits the sample on a small area of the inner surface of the capillary. The sample-containing capillaries are then placed in a reaction vessel 25 made from a commercially available vacuum hydrolysis tube. 26 The vacuum hydrolysis tube is used in a horizontal position with indentations added to support the capillaries lying horizontally (so that their ends do not contact the tube surface) and with the addition of a reagent well. The reagent (ca. 200 ~1) is added to the well and the sample-containing tubes inserted into the vessel. After cooling the reagent, the vessel is evacuated, sealed, and then placed in an oven at the appropriate temperature. After the reaction period, the reagent in the well is cooled again and the excess vapor removed by evaporation. Multiple derivatizations can be carried out by placing the sample capillaries in successive vessels containing different reagents. Following derivatization the sample tubes can be directly transferred to a Caplan and Cronin27-type gas chromatograph injector for GC/MS analysis or dissolved in a small volume of solvent for probe analysis. Transfers of very small volumes of solution from these 22 E. C. Homing and B. F. Maume, J. Chromatatogr. Sci. 7, 411 (1969). 23 j. E. Vath, M. Zollinger, and K. Biemann, Fresenius Z. Anal. Chem. 331, 248 (1988). 24 K. Biemann, this volume [18]. 25 C. E. Costello and J. E. Vath, this volume [40]. 26 Pierce Chemical Co., Rockford, IL. 27 p. j. Caplan and D. A. Cronin, J. Chromatogr. 267, 19 (1983).
324
GENERAL TECHNIQUES
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tubes can be carried out with a microliter syringe with a fused-silica capillary "needle," such as is used for on-column injections in capillary gas chromatogrpahy. Derivatization reactions which have been carried out by this method include trifluoroacetylation, acetylation, esterification (of a peptide via an intermediate azalactone formed with acetic anhydride), quaternization of amino groups, borane reduction of peptides and gangliosides, and trimethylsilylation. 23 A variety of other reactions should also be amenable to this approach.
On-Probe Derivatization in FAB Analysis For mass spectrometric analysis by FAB techniques, derivatization reactions may also be carried out directly on the sample in the liquid matrix on the probe. Examples of such reactions include acetylation, esterification, disulfide reduction, and oxidations where the reagents are added directly to the sample matrix. In some cases, the matrix itself can serve as a derivatization reagent, i.e., the reduction of disulfide bonds in a thioglycerol matrix. Using on-probe derivatization, it is possible to obtain spectra of a compound and one or more derivatives from the same sample loading on the probe. Desirable properties in a derivatization reagent for this purpose are rapid reaction at ambient temperature, relative insensitivity to atmospheric oxygen and moisture, and volatility for removal of excess reagent. Excess reagents can be removed by evaporation in the vacuum lock during probe insertion or, to protect the mass spectrometer from excessive exposure to reagents, in a separately pumped vacuum system.
Derivatization Methods The following is a sampling of general-purpose derivatization reaction procedures. These methods have been generalized from a collection of methods optimized for specific sample types, 3 from recent literature, and from collected "recipes" whose original sources are difficult to determine. In most cases, exact quantities are not stated. The reagents are generally used in large excess but on an analytical scale (e.g., nano- to picomole) even microliter quantities of reagents usually represent a large excess. The user should, however, check that the reagent amount used is in excess of the amount of sample being derivatized, Different compound types will require specifically optimized conditions for optimum derivative yields, especially for high-sensitivity quantitation; for these specific conditions
[15]
CHEMICAL DERIVATIZATION FOR MASS SPECTROMETRY
325
the reader is referred to later chapters in this volume and to the previously referenced sources. 2'3 The methods given here should be useful for qualitative work and as a starting point to develop optimized methods.
Methyl Ester Formation A solution of 2 N methanolic HCI is prepared by adding dropwise 150/.d of acetyl chloride to 1 ml of methanol. The acetyl chloride is conveniently measured using a disposable glass micropipette and added to the methanol in a glass test tube with mixing on a Vortex mixer. The solution is allowed to stand at room temperature for 5-10 rain and then added to the dried sample. After standing 1-2 hr at room temperature (a much shorter time may be adequate), the excess reagent is removed by evaporation under an inert gas stream or under vacuum. The esterification reagent should be freshly prepared; old solutions may contain chloromethane which could cause extraneous reactions. Methyl esters can also be prepared using diazomethane. This method avoids the risk of methanolysis of susceptible functional groups (e.g., amide groups in peptides). Diazomethane is highly toxic and explosive but can be handled safely in small quantities in a hood. Small quantities (less than 1 mmol) for derivatization can be prepared using a generator as described by Fales et ai.28(now commercially available) 26,29'3°which avoids the inconvenience and hazards of distillation. The diazomethane is produced by adding alkali via a syringe port in the generator to an aqueous solution of N-methyl-N-nitroso-N'-nitroguanidine (MNNG) contained in the inner of two concentric reservoirs (the more common Diazald precursor should not be used because it requires heating). The generated diazomethane dissolves in diethyl ether contained in the outer reservoir. Diazoethane can be prepared in the same manner using the homologous precursor. Deuterodiazomethane (which yields [2H2]methyl derivatives) can be prepared from MNNG by substituting 2H-labeled (-O2H) 2-(2-ethoxyethoxy)ethanol for the water and sodium deuteroxide as alkali. The derivatization is carried out by dissolving the sample in the ether solution of diazomethane. If necessary, methanol can be used to dissolve the sample. The reaction with carboxyl groups is very rapid and the persistence of the yellow color of diazomethane indicates excess reagent. For larger sample sizes, the by-product nitrogen can be observed as bubbles. The excess reagent and solvent are removed by evaporation (in a 28 H. M. Fales, T. M. Jaouni, and J. F. Babashak, Anal. Chem. 45, 2302 (1973). 29 Wheaton, Millville, NJ. 30 Aldrich Chemical Co., Milwaukee, WI.
326
GENERAL TECHNIQUES
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hood). Diazomethane can also react with carbonyl groups and double bonds to give extraneous products.
Acetylation Acetylations of OH (both alcohol and phenol) and NH (amine) groups can be carried out using acetic anhydride with either base or acid catalysis. Common reagents include 1 : 1 (v/v) acetic anhydride/pyridine or acetic anhydride/acetic acid. Dimethylaminopyridine, a much stronger catalyst than pyridine, can be used in much smaller amounts (e.g., 2% in acetic anhydride). Reactions are usually complete in 30-60 min (often less) at room temperature or with only mild heating. Peptide amino groups can be acetylated in a few minutes at room temperature with an equal volume mixture of acetic anhydride and methanol. Acetylated peaks can be identified as M,M + 3 doublets by use of 1:1 acetic anhydride/[2H6]acetic anhydride.
Trifluoroacetylation Trifluoroacetyl derivatives are commonly prepared from OH and NH groups; the amide derivatives are, in general, more stable than the ester derivatives. Trifluoracetic anhydride. (TFAA), trifluoroacetylimidazole (TFAI), or N-methylbis(trifluoroacetamide) (MBTFA) can be used as reagents. TFAA is used with either acidic (typically TFA) or basic (typically trimethylamine) catalysis. Both TFAA and TFAI methods yield nonvolatile by-products which normally require isolation of the derivative by solvent extraction. The most convenient general-purpose reagent is MBTFA. The sample is dissolved in the reagent or in a mixture (1 : 1, v/v) of the reagent and pyridine and heated at 60° for 1 hr or more. For GC/MS analysis, the mixture can be injected directly since the by-product is the innocuous and volatile N-methyltrifluoroacetamide. Alternatively, the excess reagent and by-product can be evaporated under vacuum to isolate the derivative (as long as the derivative is less volatile than the byproduct). Methyl trifluoroacetate can be used to trifluoroacetylate amino groups (e.g., in peptide methyl esters) under mild conditions) ~ The sample is dissolved in an equal volume mixture of methanol and methyl trifluoroacetate. Triethylamine is added to adjust the pH to above 8 (checked with moistened pH paper) and the solution allowed to stand at room temperature overnight. The excess reagents can be removed by evaporation. 31 S. A. Carr, W. C. Herlihy, and K. Biemann, Biomed. Mass Spectrom. 8, 51 (1981).
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Trimethylsilylation A variety of reagents are available for trimethylsilylation. A good general-purpose reagent which will silylate alcohols, phenols, amines, carboxylic acids, and thiols is N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA). The by-products trimethylsilyltrifluoroacetamide and trifluoroacetamide are quite volatile, as is the reagent itself. The sample to be silylated is dissolved in the reagent or, if necessary, in silylation-grade acetonitrile, dimethylformamide, or pyridine followed by addition of excess reagent. The reaction often proceeds well at room temperature but may require heating. A more powerful reagent contains 1% trimethylchlorosilane (TMCS) in BSTFA. For GC/MS analysis, the derivatization mixture is normally injected directly.
tert-Butyldimethylsilylation The most convenient reagent for forming tert-butyldimethylsilyl (TBDMS) derivatives is N-methyl-N-(tert-butyldimethylsilyl)trifluoroacetamide (MTBSTFA). Its use is similar to BSTFA but often requires more vigorous reaction conditions to transfer the bulkier silyl group. MTBSTFA containing 1% of the corresponding chlorosilane (available commercially)32is probably the best first choice for most compounds. The TBDMS group is much more stable toward hydrolysis than the TMS group; unlike TMS derivatives, the TBDMS derivatives can be isolated, if desired, for direct probe analysis.
Methoxime Formation Methoxime derivatives are used to reduce the polarity and hydrogen bond acceptor potential of aldehyde and ketone carbonyl groups. A disadvantage of the derivative is the possible formation of chromatographically separable syn and anti products due to restricted rotation about the methoxime double bond. The derivative is prepared by adding a 2% solution of methoxyamine hydrochloride in pyridine to the sample. Reaction conditions vary for different compounds but are typically several hours to overnight at room temperature or one to a few hours at 60°. The excess reagent is not volatile, therefore the derivatized sample is usually recovered by adding water (or aqueous NaCI) and extracting with ethyl acetate. In a multistep derivatization involving water-sensitive derivatives, the methoximation must be performed first. 32 Regis Chemical Company, Morton Grove, IL.
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On-Probe Derivatization Methods On-probe derivatization methods are commonly used in FAB analysis, but can be carried out on sample in the probe capillary in EI and CI probe analysis. Thus, reaction conditions are less stringent than for conventional derivatizations. The following listed below are commonly used on-probe procedures. Methyl Ester Formation. One microliter of 2N methanolic HC1 is added to the sample (or in the case of FAB, the sample/matrix mixture) on the probe tip and allowed to stand for a few minutes at room temperature. The sample is then reinserted into the vacuum lock and the excess reagents pumped away. If the ester peaks are not observed, the derivatization can be repeated for a longer period. Acetylation of Amino Groups in Peptides. A 0.5-/zl aliquot of 1:1 (v/v) methanol/acetic anhydride and 0.5/zl of pyridine are added to the sample on the probe tip and allowed to stand at room temperature for a few minutes. The excess reagents are then removed in the vacuum lock prior to analysis. Reduction of Disulfide Bonds in Peptides. Raising the pH of a sample in thioglycerol or dithiothreitol (DTT)/dithioerythritol (DTE) matrix by addition of 0.5/zl 28% aqueous ammonia will promote reduction by the thiol matrix. Alternatively, 1/zl of either 1 : 1 (v/v) 0.2 M N-ethylmorpholine buffer (pH 8.5)/I M DTT or 1:1 (v/v) aqueous ammonia (28%)/ thioglycerol is added and allowed to stand for a few minutes at room temperature. 33 The sample is then acidified with HCI or trifluoroacetic acid, and FAB matrix added if necessary, prior to removal of excess reagent in the vacuum lock and analysis. Performic Acid Oxidation. Pefformic acid is prepared by adding 5% (by volume) of 30% hydrogen peroxide to 99% formic acid and allowing the solution to stand at room temperature for 2 hr. One microliter of the solution is added to the sample in glycerol matrix and allowed to stand for several minutes prior to analysis. Summary and Future Prospects Chemical derivatization in relation to mass spectrometry has evolved from primarily volatility considerations to a primary emphasis upon manipulation of the ion chemistry in the mass spectrometer to increase the information yield. The advent of FAB methods at first appeared to obviate the need for derivatization, but now it is recognized that FAB presents 33 H. Rodriqucz, B. Nevins, and J. Chakcl, in "Techniques in Protein Chemistry" (T. E. Hugli, ed.), p. 186. Academic Press, San Diego, 1989.
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new opportunities to exploit derivatization. Likewise, the rapidly developing field of tandem mass spectrometry creates additional opportunities for the creative use of chemistry in conjunction with mass spectrometry to increase the ability to solve structural problems. In the area of protein structure, for example, mass spectrometry has already established itself as the "third leg of the stool" of protein primary structure methods (along with Edman and DNA sequencing). To date, however, mass spectrometry has been little exploited for other than determination of primary covalent structure. In conjuction with creative uses of chemical modifications (derivatizations), mass spectrometry offers the potential to yield three-dimensional information as well. Chemical linkage experiments can yield distance geometry measurements which, in conjunction with computer molecular graphics, can be used to refine three-dimensional structures. As a further extension, chemical trapping of intermediate states offers the potential to gain information on molecular dynamics. Thus, there appears to be a wide range of future applications for chemical derivatization in the broadest sense of the term. Just as with protein chemistry, whose demise was prematurely announced some years ago, 34 chemical derivatization in relation to mass spectrometry is far from dead. 34 A. D. B. Malcolm,
Nature (London) 275,
90 (1978).
[16] I n t r o d u c t i o n o f D e u t e r i u m b y E x c h a n g e for Measurement by Mass Spectrometry By
JAMES A. MCCLOSKEY
The chemical incorporation of deuterium followed by measurement of the intact product by mass spectrometry has for a number of years played a major role in three, often overlapping areas: the structural characterization of molecules of unknown structure; to gain information on the mechanisms of chemical or biological reactions; and as an aid in the interpretation of mass spectra. 1 In each of these areas mass spectrometry offers several general advantages: (1) relatively routine measurements can be made in the sample quantity range 10-11-10-6 g, sometimes in mixtures or without extensive sample purification; (2) the sites of labeling can often be deterI H. Budzikiewicz, C. Djerassi, and D. H. Williams, " S t r u c t u r e Elucidation of Natural Products by M a s s S p e c t r o m e t r y , " Vol. 1, Alkaloids, Chap. 2. Holden-Day, San Francisco, 1964.
METHODS IN ENZYMOLOGY, VOL. 193
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