Mass Spectrometry of Organophosphorus Compounds

BY J. R. CHAPMAN

1 Introduction The history of the application of mass spectrometry t o the analysis of organo- phosphorus compounds provides many illustrations of the shortcomings of conventional electron-impact ionization and the advantages t o be gained from newer ionization techniques. In the area of synthetic chemistry, electron- impact ionization has been widely used for the analysis of phosphorus compounds and has often been able t o provide considerable structural information. However, a wide range of compounds based o n the oxyacids of phosphorus undergo extensive fragmentation under electron-impact condi- tions, so that the percentage of ion current that is carried by the molecular and other high-mass ions is very low, making complete identification difficult. Ionic compounds such as phosphonium salts are not amenable t o analysis under electron-impact conditions. Thus, electron-impact ionization on its own is a less than suitable method for over half the compounds recorded in synthetic phosphorus chemistry. Chemical ionization and, t o a lesser extent, field desorption are now increasingly used as ionization techniques in this field.

In the field of natural products, the presence of ionic phosphate groups and the larger molecular size of these materials, together with the presence of more labile sugar moieties in some cases, has underlined shortcomings in both mass-spectrometric ionization and the techniques for introducing a sample into the spectrometer. Thus, earlier applications of mass spectrometry t o nucleoride analysis employed structural elucidation based on the fragmenta- tion that is induced by a combination of electron-impact and pyrolytic techniques. Alternatively, chemical degradation or derivatization techniques had t o be used prior t o mass-spectrometric analysis. However, the application of techniques such as field desorption and, more recently, energetic- particle bombardment, has provided a means by which the direct analysis of these natural products may be simply achieved.

Because of the considerable importance of ionization techniques in the analysis of organophosphorus materials, Section 2 of this Report is devoted to a more detailed discussion of their availability, applications, and limitations.

2 Techniques Chemical Ionization and ‘In- Beam’ Evaporation Techniques.-Under standard electron-impact (EI) conditions, the interaction in the ion source of

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Muss Spectrometry of Organophosphorus Compounds 279

energetic electrons with sample molecules that have ionization potentials of 7-1OeV produces molecular ions that contain a considerable amount of excess energy. This energy is largely dissipated through fragmentation processes. If the ion source now also contains a high pressure (0.1 - 1 Torr) of a reagent gas such as methane, so that the ratio of reagent gas to sample is in the order of 1 O 3 or 1 O4 t o 1, then the principal process carried out by the electrons becomes the ionization of the reagent gas. In the case of methane, ionization followed by ion-molecule reactions eventually produces two dominant ionic species, CHS+ and C2Hs+. Both of these reagent ion species can now act to ionize the sample molecules present in the source. This is chemical ionization (CI).' A major reaction with methane reagent gas is ionization by proton transfer [reaction ( 1 ) I .

M + CH,' + MH+ + CH, (1)

Proton transfer is a considerably less energetic process than ionization by electron impact, with the overall result that whilst some fragmentation does occur, the percentage of the ion current that is carried by the so-called quasi- molecular ion MH+ is generally much greater than that carried by the molecular ion M+, formed under electron-impact conditions. The spectra of parathion as obtained by EI and CI are given in Table 1 for comparison.

Table 1 Major ions in the CI spectrum (using methane)2 and the El spectrum o f parathion (mol. wt=291)

CI EI

mlz Rel. Composition mlz Rel. mlz R el. in t. in 1. in t.

292 100 (M + H)' 6 3 36 97 100 320 19 (M +C2H5)+ 6 4 21 109 96

65 5 0 125 33 75 15 137 41 76 1 1 139 36 81 25 155 26 93 20 29 1 15

A further advantage of chemical ionization is that, by the use of different reagent gases, reagent ions of differing reactivity may be produced. For example, the use of isobutane as the reagent gas produces a t-butyl ion as the principal reagent ion. As this ion is a considerably weaker proton donor than the CHS+ ion, isobutane can be used to form quasi-molecular ions which undergo much less fragmentation. Still milder ionization conditions may be achieved by the use of ammonia, which provides protonated molecular ions MH+ or adduct ions (M +NH4)+, depending on the basicity of the sample r n o l e ~ u l e . ~ Such milder reagent gases are also more selective, so that, for example, saturated hydrocarbons will not be ionized by ammonia. ' M. S. B. Munson and F. H. Field, J. Am. Chem. SOC., 1966, 88, 2621.

R. L. Holmstead and J. E. Casida, J. Assoc. Off Anal. Chem., 1974, 5 7 , 1050. J . M . Desmarchelier, D. A. Wustner, and T. R. Fukuto, Residue Rev., 1976, 6 3 , 77. T. Keough and A. H. DeStefano, Org. Mass Spectrom., 1981, 16, 527.

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28 0 Organ o p h 0 s p h or us Ch e rnis try

The use of a very mild reagent gas, giving little or no fragmentation, can be particularly advantageous when the detection of a particular compound by monitoring a relatively intense specific ion is t o be accomplished. The use of a gas that gives more fragmentation or the complementary use of electron- impact ionization may be preferred where more information, for complete structural analysis, is required. More detailed applications of the technique of chemical ionization will be found in Sections 3 and 4.

The use of chemical ionization does not itself remove the necessity for vaporization of the sample, either from a conventional heated probe that is introduced into the ion source or on injection into a coupled gas chromato- graph. Obviously, many materials are sufficiently thermolabile that decomposition occurs during evaporation. The use of modified techniques for evaporation of the sample can be helpful in these cases.

All of these modified techniqes involve evaporation of the sample very close t o the point of ionization, and therefore may be spoken of as 'in-beam' techniques. In many cases, high rates of heating (2 10 "C s- ') have been used, so that evaporation takes precedence over thermal d e c o m p ~ s i t i o n . ~ Evapora- tion of the sample from an inert support material, such as gold6 or a polyimide-coated wire,7 is an important feature in many cases.

Instrument manufacturers provide facilities for these specialized evapora- tion techniques under the acronym DCI (direct or desorption chemical ionization). Rapid in-beam evaporation is also a viable technique under EI conditions (see p. 288). Negative-ion Chemical Ionization.-Until recently, analysis of negative ions was little used in mass spectrometry. This was principally because the negative ions that are produced under the conditions of electron-impact ionization are those corresponding t o structurally uninformative low-mass fragments, and even these are produced only in low yield. More useful spectra can be produced by dissociative electron-capture processes, using a less energetic electron beam, but, even in these cases, information on molecular weights is absent and the sensitivity is low.

An impetus t o the analysis of negative ions was provided by the realization that a reagent gas for chemical ionization, e.g. methane, interacts with a primary beam of energetic electrons t o provide not only positive reagent ions but also a large population of secondary electrons, having only thermal energy [reaction (2)] . These electrons may be captured by sample molecules that have a positive electron affinity, in a non-dissociative process, t o provide molecular anions M-. This process, commonly referred to as negative-ion chemical ionization, has the advantages of being selective for electron- capturing compounds, providing an extremely mild form of ionization, and achieving high sensitivity.8 In certain cases, electron-capture ionization can

* G. B. Daves, Jr.,Acc. Chem. Res., 1979, 12, 359. E. Constantin, Y . Nakatini, G. Ourisson, R. Hueber, and G. Teller, Tetrahedron Let t . , 1980,21,4745. V . H . Reinhold and S. A . Carr, Anal. Chem., 1982, 54, 499. D. F. Hunt, G . C. Stafford, Jr . , F . W. Crow, and J . W. Russell, Anal. Chem., 1976, 48, 2098.

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Mass Spectrometry of Organophosphorus Compounds 28 1

offer sensitivity that is higher by orders of magnitude than positive-ion chemical ionization.

In addition to electron-capture ionization, negative-ion operation provides the possibility of ionization through ion-molecule reactions, in a manner analogous to the positive-ionization methods described earlier. An important anionic reagent ion is C1-, generated from a number of alternative sources, such as carbon tetrachloride, d ich l~romethane ,~ or dichlorodifluoromethane (Freon 12)."

The C1- ion reacts principally by the formation of (M+Cl)- adduct ions. Not only is the chloride ion a very mild CI reagent, producing virtually no fragmentation, it is also relatively selective. Thus, C1- reacts with a wide range of phosphate esters to give (M +C1)- ions l 1 but will not, for example, react with hydrocarbons. The direct analysis of a lubricating oil that contains a zinc dithiophosphate ester by this method produces a chloride-attachment spectrum of the phosphate ester, with virtually no interference from the hydrocarbons and no need for prior purification of the sample.'* Field Desorption and Ionization by Energetic Particles.-A field-desorption (FD) source contains an emitter - a tungsten wire of diameter 1 0 p m , on which microscopic carbonaceous dendrites have been grown - which is held at a high positive potential with respect t o an adjacent counter-electrode. The sample is deposited on this emitter from solution. Positive sample ions, formed on the surface of the emitter in the intense electric field, are drawn towards the counter-electrode and analysed. The emitter wire is heated during analysis, to promote diffusion and volatilization of the sample. These processes are also assisted by the structure of the dendrites, which allows the sample to be distributed over a very large surface area. No evaporation (to achieve a given vapour pressure of the sample) prior t o ionization is required, unlike the introduction of sample from a probe or from a gas chromatograph prior to EI or CI.

Despite being a relatively difficult technique experimentally, field desorp- tion has been widely and successfully used in the analysis of involatile and labile materials.13 Field desorption is a very mild ionization process, so that the spectra show intense M+ and/or (M+H)+ ions, with the possibility of increasingly abundant fragment ions as the temperature of the emitter is raised. In the field of phosphorus compounds, the technique has particularly been applied to the analysis of nucleotides and phospholipids, which is discussed in more detail below.

Ionization by energetic particles embraces a range of ionization techniques which have come to prominence more recently. Essentially, the sample is

H. Y. Tannenbaum, J. D. Roberts, and R. C. Dougherty, Anal. Chem., 1975,47, 49. l o A. K. Ganguly, N . F. Cappuccino, H. Fujiwara, and A. K. Bose, J . Chem. SOC., Chem.

Commun., 1979, 148. I ' R. C. Dougherty and J. D. Wander, Biomed. Mass Spectrom., 1980, 7 , 401. K. P. Morgan, C. A. Gilchrist, K. R. Jennings, and 1. K. Gregor, Int. J. Mass Spectrom. Ion Phys., 1983, 46, 309. '' H.-R. Schulten, Int. J . MassSpectrom. ion Phys., 1979, 32, 97.

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28 2 Organophosphorus Chemistry

Table 2 Methods o f ionization by energetic particles

Method Typical particle Reference t ype and energy

".'Cf fission fragment ionization 100 MeV Tc' 16 Caesium ion bombardment 8-28 keV Cs' 17 Fast- atom bombardment 5 keV ArO 15 Secondary ion mass spectrometry 5 keV Ar' 14 Laser desorption 256 nm photon 18

placed on a target, which is then bombarded, using a beam of neutral atoms, ions, or photons, t o produce sample ions by a sputtering process. The different methods are mainly distinguished by the type and energy of the bombarding particles (Table 2).

The most popular method at the time of writing is fast-atom bombard- ment (FAB), which uses a beam of energetic neutral atoms. This technique, which employs a matrix material, such as glycerol, to dissolve the sample and to promote the diffusion of the sample to the surface layers, has the advantages of experimental simplicity and ready compatibility with magnetic- sector mass spectrometers which permit the analysis of ions of high mass.

The most obvious advantage of energetic-particle- bombardment techniques is their ability in many cases to provide information on molecular and fragment ions from molecules of extreme polarity and high molecular weight, well beyond the practical limits of field desorption (c f . p. 286). Additionally, a technique such as FAB, when compared with FD, is simpler, operates with equal facility in the negative- and positive-ion modes, is more sensitive, and is less affected by the presence of impurities. Against this, FD produces far fewer background ions, particularly since it employs no matrix material, and can be a better method for the analysis of mixtures, particularly for the detection of minor components in mixures (cf. p. 288). Identification of Metastable Ions.-Although the electron-impact spectrum of a phospholipid usually shows no molecular ion, it was demonstrated quite early on that intact molecular ions are formed in the source under EI condi- tions but that these fragment before reaching the d e t e ~ t o r . ' ~ These so-called 'metastable' ions may be identified, using a variety of experimental techniques that are available on double-focusing magnetic-sector instruments.

If a metastable ion of mass m l fragments as shown in reaction (3) , then techniques which make use of the energy-analysing properties of double- focusing instruments may be used to elucidate the initial mass ml, even

l 4 A. Eicke, W. Sichtetmann, and A. Benninghoven, Org. Mass Spectrom., 1980, 15,

l 5 M. Barber, R. S. Bordoli, R . D. Sedgwick, and A. N. Tylor, Nature (London), 1981,

l6 R. D. MacFarlane and D. F. Torgerson, Science, 1976, 191, 920. l 7 W.Ens, K. G. Standing, B. T. Chait, and F. H. Field, Anal. Chem., 1981, 5 3 , 1241. lR M. A. Posthumus, P. G. Kistemaker, H. L. C. Meuzelaar, and M. C. Ten Noever de

l 9 R. A. Klein, J. Lipid Res., 1971, 12, 628 .

289.

293, 270.

Brauw, Anal. Chem., 1978, 5 0 , 985.

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Mass Spectrometry of Organophosphorus Compounds 283

though the recorded ions have mass r n 2 . * O Thus, molecular weights may be elucidated in certain cases, even though the molecular ion is absent from the normal spectra.lg,

m l * + + m2*+ + m 3 (3)

Another technique for metastable ions, known as ‘linked scanning’,22 may be used to record selectively only those ions that are produced by subsequent collision-induced dissociation of a chosen molecular ion that has been formed by a soft ionization technique such as field desorption23 ( c f . p. 285). This method provides an alternative to thermolysis as a method of increasing the amount of information on fragment ions that is available from soft ionization techniques. Liquid Chromatography.-Combined gas chromatography-mass spectro- metry (g.c.-m.s.) is a well-established technique. However, since gas chroma- tography is limited in its ability t o handle polar and labile materials, there is an obvious interest in combined liquid chromatography-mass spectrometry (1.c.-ms.) in the field of phosphorus chemistry, as in many others.

The 1.c.-m.s. interfacing methods that are currently available are the moving belt, in which a mechanical transport system is used to convey the sample from the column to the ion source,24 and the direct liquid-introduc- tion (DLI) system, in which the liquid eluent itself is used as the transport medium.25 Although a preference for the DLI system seems to be emerging, 1.c.-m.s. interfacing is still an evolving technique, and not yet at the stage where definitive comparisons of the available systems may be made. It is more practicable at this stage merely to draw the reader’s attention to published applications in the field of phosphorus chemistry, viz. analysis of nucleotides by thermospray (DLI) 1.c.-m.s. ,26 analysis of phospholipids (DLI),27 and analysis of organophosphorus pesticides (DLI).28

3 Natural Products Nuc1eotides.-The principal mass-spectrometric techniques that have been applied to the analysis of nucleotides are electron-impact ionization, field desorption, and (more recently) ionization by energetic particles.

Electron-impact ionization has been used in the analysis of oligonucleotides despite its known inability to offer information on molecular weights by direct analysis of the nucleotides. The application of this technique has been

2 o R. G. Cooks, J . H. Beynon, R. M . Caprioli, and G. R. Lester, ‘Metastable Ions’,

21 S. G. Batrakov, V. L. Sadovskaya, V. N. Galyashin, B. V. Rosynov, and L. D.

22 A. P. Bruins, K. R. Jennings, and S. Evans, Znt. J. Mass Spectrom. Ion Phys., 1978,

23 K. M. Straub and A. L. Burlingame, Adw. Spectrom., 1980, 8, 1127. 24 W. H. McFadden, J. Chromatogr. Sci., 1979, 17, 2. 2 5 P. J. Arpino and G. Guiochon, Anal. Chem., 1979, 51, 682A. *‘ C. R. Blakley, J . J . Carmody, and M . L. Vestal, J. Am. Chem. SOC., 1980, 102, 5931. 21 F. R. Sugnaux and C. Djerassi, J. Chromatogr., 1982, 251, 189. 2 R C. E. Parker, C. A. Haney, and J . R. Hass, J. Chromatogr., 1982, 2 3 7 , 233.

Elsevier, Amsterdam, 1973.

Bergel’son, Bioorg. Khim., 1978, 4, 1390.

26, 395.

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284 Organophosphorus Chemistry

mainly due t o the efforts of W i e b e r ~ , ~ ~ - ~ ~ who has developed methods for the interpretation of information from fragment ions in the spectra of oligonucleo- tides. These spectra, which are produced partly by electron-impact-induced processes and partly by thermolysis, offer information on the bases that are present and (in smaller oligonucleotides) their sequence 3 2 , 33 as well as on the location and identity of protecting groups that have been introduced during synthetic procedure^.^^ This technique is a particularly sensitive method for confirming that all blocking groups have been removed following synthesis.

Another application that has successfully been examined by Wiebers is the detection and identification of abnormal nucleotide residues in DNA.M For example, this method has been used to establish the occurrence of 5-methyl- and 5-hydroxymethyl-cytidine. Wiebers’ original work on the identification of bases has been continued and developed by other author^.^' A more recent report by Ulrich and co-workers also discusses the pyrolysis-electron-impact mass spectrometry of protected phosphotriester oligodeoxyribonucleotides.36 The recorded spectra give ions that are characteristic of the bases and the protecting groups.

An alternative approach to the analysis of nucleotides, using electron- impact ionization, has been via the formation of derivatives. Both Baker 37

and, more recently, Pettit 38 have used trimethylanilinium hydroxide t o permethylate nucleoside monophosphates, for subsequent analysis by g.c.- m.s. Both authors used electrons of lower energy (1 1-20 eV), and Pettit reported characteristic molecular and fragment ions from a number of samples. The 2’-, 3’-, and 5‘-monophosphates of adenosine all gave distinct spectra as their hexamethyl derivatives, although the method was not completely satisfactory for the unequivocal location of the phosphate

Thymidine and uridine phosphates were also analysed, the latter also by field ionization, when only a molecular ion was observed.38

Ribonucleosides that are liberated by hydrolysis with a phosphatase can be permethylated prior t o g.c.-m.s., using sodium methoxide or methyl iodide in DMSO. In this case the concentration of the quasi-molecular ion that is formed by chemical ionization (using isobutane) was monitored, as the basis of a quantitative method.39

Trimethylsilylation of halogenated nucleotides and nucleosides has been used by Gelpi t o permit their analysis by g.c.-m.s. techniques. Finn 41 has

2 9 M . A. Armbruster and J. L . Wiebers, Anal. Biochem., 1977, 83, 570. 30 J. L. Wiebers,Anal. Biochem., 1973, 51 , 542. 3 ’ D. K . Burgard, S. P. Perone, and J . L. Wiebers, Anal. Chem., 1977, 49, 1444. 32 J . L. Wiebers and J . A. Shapiro, Biochemistry, 1977, 16, 1044.

34 J. L. Wiebers, Nucleic Acids Res., 1976, 3, 2959. D. K . Burgard, S . P. Perone, and J. L. Wiebers, Biochemistry, 1977, 16, 1051.

N. Turkkan, F. Soler, and K. Janowski, Biomed. Mass Spectrom., 1982, 9 , 9 1. J . Ulrich and M . J . Bobenrieth, Z . Naturforsch., Tril. B, 1980, 35, 212.

37 K. M . Baker, Adv. Mass Spectrom. Biochem. Med.. 1976, 1, 231. 38 G. R. Pettit, J. J. Einck, and P. Brown, Biomed. Mass Spectrom., 1978, 5 , 153. 39 I . Jardine and M. M. Weidner, J . Chromatogr., 1980, 182, 395. 40 E. Gelpi, J . Pares, and C. Cuchillo, Adv. Mass Spectrom. Biochem. Med., 1976, 1,

41 C. Finn, H. J. Schwandt, and W. Sadee, Biomed. Mass Spectrom., 1979, 6, 194.

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Mass Spectrometry o f Organophosphorus Compounds 28 5

trimethylsilylated the free bases that are produced by hydrolysis of a nucleo- tide, subsequently monitoring the M+ and (M - CH3)’ ions during analysis by g.c.-m.s., t o provide a method for the quantitative determination of uracil and thymine at the picomole level.

Unlike electron-impact ionization, field desorption produces spectra from nucleotides that contain peaks which give information on molecular weights. Fragment ions are also present which identify the base. Ions with a low mass in the spectrum of 5’-AMP suggest the presence of ribose rather than d e o x y r i b o ~ e . ~ ~ In the original paper, measurement of mass was used to confirm the composition of each peak.42 Extension of this work to dinucleo- tide monophosphates produced spectra that again gave information about the molecular weights of the compounds and the separate units from which they were made up.43 Pyrolysis-field desorption of deoxyribonucleic acid gave complex spectra which nevertheless contained information related to the constituent bases.44

In his most recent paper, Schulten4’ has used FD t o identify protected synthetic deoxyribonucleotides after their separation by high-performance liquid chromatography (h.p.1.c.). The spectra contained intense cationized molecular ions and a small number of structurally significant fragment ions, as well as information on organic and inorganic impurities. H.p.1.c. can offer a valuable method of cleaning up samples prior t o F D ( c f . p. 282), since the performance of a field desorption source is very much dependent on the presence or absence of impurities. This problem, and that of deterioration of the emitter in the analysis of nucleotides, has been discussed by Budzikiewicz and co-workers.& These same authors used FD to study the course of the reaction of several dinucleoside phosphates with diazo- methane.47

In a particularly elegant study, Burlingame 23 has applied field desorption- collision-induced dissociation (FD-CID) mass spectrometry to the sequencing of polynucleotides that have been modified by interaction with intermediates derived from the potent carcinogen benzo [alpyrene. In this technique, collision with inert gas molecules is used to enhance the fragmentation of field-desorbed molecular ions, These collision-induced fragment ions are then selectively recorded, using the linked-scan technique mentioned on p. 283. By this means, the direct analysis of a modified deoxyadenosine-deoxy- thymidine dinucleoside was achieved.

The FD spectra of cyclic nucleotides have also been reported.48 Cyclic AMP has also proved amenable to DCI analysis, giving information on

4 2 H.-R. Schulten and H. D. Beckey, Org. MassSpectrom., 1972, 7, 861.

4 4 H.-R. Schulten, H. D. Beckey, A. J . H . Boerboom, and H. L. C. Meuzelaar, Anal.

4 5 H. M. Schiebel and H.-R. Schulten, Z. Naturforsch., Teil. B, 1981, 36, 967. 46 H. Budzikiewicz and M. Linscheid, Biomed. Mass Spectrom., 1977, 4. 103. 4 7 M . Linscheid, G. Feistner, and H. Budzikiewicz, Zsr. J . Chem., 1978, 17, 163. 48 C.-H. Wang, Y.-X. Wang, K.-X. Cao, L.-H. Chang, L.-T. Ma and X. Wang, Huah

H.-R. Schulten and H. M. Schiebel, Fresenius’ 2. Anal. Chem., 1976, 280, 861.

Chem., 1973,45, 2358.

4 3

Hsueh Tung Pao, 1981, No. 1, p. 11.

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286 Organophosphorus Chemistry

molecular and fragment ions, using methane 49 or ammonia ’” as the reagent gas. FD cannot be used t o distinguish isomeric nucleoside 3’- and 5’-mono- phosphates whereas the EI spectra of the per- trimethylsilyl derivatives of these compounds do allow a distinction t o be made.”

The analysis and sequencing of protected oligonucleotides, using an energetic-particle-ionization method, was first reported by McNeal and co- workers in 1980,52 using the method involving a 252Cf source (see Table 2) that had been developed by McFarlane and Torgerson.I6 This technique, although very simple in practice, has been used by few investigators because of problems in handling the source material and because of the specialized nature of the mass spectrometer used. It has, however, provided results which remain a target for other more accessible energetic-particle techniques.

In a series of papers, McNeal and co-workers have recorded molecular and dimer ions from a protected synthetic deoxydodecanucleotide of molecular weight 6275 53 and have demonstrated the use of the technique in sequencing protected ribo-oligonucleotides that contain up t o seven nucleoside units. 54-56

Information giving the molecular weight is available in the positive-ion mode whilst the negative-ion spectra contain two nested series of fragment ions which resemble the summed products of two parallel enzymatic digests, utilizing a 5’- and a 3’-exonuclease. The information on base sequence from one set of fragment ions is thus mirrored in the complementary set.

Ionization of fully protected di- and tri-ribonucleoside phosphates with Cs+ ions (of energy 8-28 kV) has produced similar data.57 The positive- and negative-ion spectra again contain information enabling the determination of the molecular weight, the identification of the protecting groups and bases present, and the determination of the base sequence (the last, again, particu- larly from the negative-ion spectrum).

Ionization using sources of less energetic particles has so far been restricted t o unprotected nucleotides. Thus, mass spectra from mono- and di-nucleotides have been produced by the impact of low-energy ions l4 and of low-energy neutral atoms l5 (molecular SIMS and fast-atom bombardment, respectively) and an (M - H)- ion from a tetranucleoside triphosphate has been observed at m/z 1172, using the FAB technique.” Spectra obtained by using these tech-

49 D. F. Hunt, J . Shabanowitz, F. K. Botz , and D. A. Brent, Anal. Chem., 1977, 49, 1160. E. J . Esmans, E. J . Freyne, J . H. Vanbroeckhoven, and E’. C. Aldenveireldt, Biomed. Mass Spectrom., 1980, 7 , 377. H. Budzikiewicz and G . E‘eistner, Biomed. Mass Spectrom., 1978, 5 , 5 12.

5 2 C. J . McNeal, S. A. Narang, R. D. Macfarlane, H. M. Hsiung, and R. Brousseau, Proc.

53 C. J . McNeal and R . D. Macfarlane,J. Am. Chem. SOC., 1981, 103, 1609. 54 C. J . McNeal, K. K. Ogilvir, N. Y . Theriault, and M . J . Nemer, J. Am. Chem. SOC.,

1982, 104, 972. 5 5 C. J . McNeal, K. K. Ogilvie, N. Y . Theriault, and M. J . Nemer, J. Am. Chem. SOC.,

1982,104,976. 56 C. J . McNeal, K. K. Ogilvie, N . Y . Theriault, and M. J . Nemer, J. Am. Chem. SOC.,

1982, 1 0 4 , 9 8 1 . W . Ens. K. G. Standing, J . B. Westmore, K. K. Ogilvie, and M. J . Nemer, Anal. Chem., 1982, 54, 960. D. H. Williams, C. Bradley, G. Bojesen, S. Santikarn, and L. 2. E. Taylor, J. Am. Chem. SOC., 1981, 103, 5700.

Natl. Acad. Sci. USA, 1980, 7 7 , 735.

57

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Mass Spectrometry of Organophosphorus Compounds 287

niques are very similar to those observed when using the sources of more energetic particles, and it is therefore expected that techniques such as FAB should also be applicable to the analysis of protected nucleotides. Somewhat similar spectra to those obtained by using less energetic particles have been recorded for unprotected mono- and di-nucleotides by workers who used laser-desorption ionization techniques.I8

Nucleotides have recently become amenable to analysis by 1.c.-m.s., using a technique for direct introduction of a liquid that has been developed by Vestal and co-workers.26 In this method, ions that have been pre-formed in solution can be evaporated from the surface of tiny droplets, these being formed when the flow from the liquid chromatograph is sprayed into the vacuum system of the mass spectrometer. Spectra with intense quasi- molecular ions and excellent signal- to-noise characteristics have been obtained for adenosine monophosphate and two dinucleoside monophos- phates by using this mode of operation. Phospholipids.-Phospholipids are another class of polar compounds that present difficulties in analysis. Many routine analytical methods rely on hydrolytic procedures, after which the component fatty acids may be deter- mined by chromatographic techniques. A more direct method of analysis is, however, provided by the use of field desorption.

In an early paper," Wood and co-workers reported on the problems caused by organic and inorganic contaminants in the FD analysis of phospho- lipids. In particular, inorganic ions such as Na" result in low ion intensities, increased fragmentation, and the formation of cluster ions or even complete masking of the spectra of the lipids. More recently, this same school reported on the successful use of a modified extraction technique to clean up phospho- lipid samples prior to FD analysis, giving much improved spectra.60 The FD spectrum of dipalmitoylphosphatidic acid that is published in this later paper gives information on the molecular weight and consistuent fatty acids. The (M + H)" ion is the base peak whilst the (M + Na)+ ion has been reduced to the level of 1% of that intensity.

Published data from field-desorption studies are now available for the following classes of phospholipids: phosphatidylcholines,5g~ phospha- tidy let ha no la mine^,^^ phosphat idic acids,59 , glycerophosphoryl lipids,59 and the aldehydogenic phospholipids (1).@ These spectra always show intense quasi-molecular ions, but structurally informative fragment ions are less common. In a very recent study, silicon emitters were used for field- desorption studies of phospholipids and related derivative^.^' 5 9 G. W. Wood, P. Y. Lau, G. Morrow, G. N. S. Rao, D. E. Schmidt, Jr., and J . Tuebner,

6 o G. W. Wood and S. E. Perkins, Anal. Biochem., 1982, 122, 368. Chem. Phys. Lipids, 1977, 18, 316.

G. W. Wood, P.-Y. Lau, G. N. Rao, and G. N. S. Rao, Biomed. Mass Spectrom., 1976, 3 , 172.

6 1

6 2 G. W. Wood, P. A. Tremblay, and M. Kates, Biomed. Mass Spectrom., 1980, 7 , 11. 6 3 G. W. Wood and P. Y. Lau, Biomed. Mass Spectrom., 1974, 1, 154. 6 4 A. V. Chebyshev, S. P. Kabanov, A. A. Perov, G. A. Serebrennikova, S. E. Kupri-

yanov, and R. P. Evstigneeva, Bioorg. Khim., 1977, 3, 1370. J . Sugatani, M . Kino, K. Saito, T. Matsuo, H. Matsuda, and I . Katakuse, Biomed. Mass Spectrom., 1982, 9, 293.

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288 Organophosphorus Chemistry

C H ~ O C H = C H R '

I CH20CH2(CH2)14Me

I I C H O C O R ~ CHOH

An interesting study of the practical use of FD in the analysis of phospho- lipid mixtures has been presented by Catlow,66 who points out that whereas conventional FD gives moiecular, fragment, and adduct ions, e.g. (M + CH3)+ and (M + Na)', from phospholipids, the addition of a strong acid such as toluene-p-sulphonic acid changes the spectrum so that it comprises only a (M+H)+ ion.67 This simplicity and the absence of interfering back- ground which make the method ideal for the analysis of mixtures may be contrasted with FAB results also obtained by Catlow on these mixtures. In this case, molecular species are also seen, but the intense background that is characteristic of FAB spectra means that minor components of mixtures are usually not recognized.

Chemical ionization may also be used t o provide spectra of intact phospho- lipids. For example, the 1 -O-alkylglyceryl-3-phosphorylcholine (2 ) (0- deacetyl platelet-activating factor) that is released from leucocytes was identified by CI mass spectrometry, using isobutane as the reagent gas6* The CI spectrum gave information on molecular weights and on fragment ions that resulted mainly from cleavages at phosphate bonds.

Sugnaux and Djerassi 27 have compared the methane and ammonia DCI spectra of dimyristoylphosphatidylcholine with the spectrum, run in the presence of ammonia, of the same sample when introduced from a liquid chromatograph via a DLI interface. Since the quality of DCI spectra varies considerably with time, these authors concluded that the 1.c.-DLI technique is better for quantitative determination of this material. The quasi-molecular ion is less intense in the DLI spectrum than in the ammonia DCI spectrum, but is still more intense than that observed in the methane DCI spectrum. Fragmentation in all three spectra results mainly from cleavage of phosphate ester bonds.

Workers at Strasbourg have demonstrated that phospholipids that are evaporated close to the electron beam from an inert gold support give spectra with information on the molecular ion even under electron-impact condi- t ions6 A stable beam, allowing scanning or mass-measurement experiments, is obtained by using this technique. Under EI conditions, fragment ions (allowing identification of the constitutent fatty acids) are also seen. Sugnaux and Djerassi 27 have also recorded the EI spectrum of dimyristoylphospha- tidylcholine when it was being rapidly evaporated.

6 7 T. Keough and A. J . DeStefano, Anal. Chem., 1981, 53, 25 . 6 8 J. Polonsky, M . Tence, P. Varenne, B. C. Das, J . Lunel, and J . Benveniske, Proc. Natl .

D. A. Catlow, Int. J. Mass Spectrom. Ion Phys., 1983,46, 387. 6 6

Acad. Sci. USA, 1980, 7 7 , 7019.

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Mass Spectrometry o f Organophosphorus Compounds 289

Under more normal conditions of introduction of the sample, when the molecular ion is not seen, information on the molecular weight has been obtained for phosphatidylglycerols, lecithins, and phosphatidylethanolamines by using one of the techniques for metastable ions that was described above (p. 283).21

G.c.-m.s. techniques can be used for the structural analysis of phospho- lipids, following their enzymatic hydrolysis. Monohydroxy-compounds that are formed on hydolysis are analysed as trimethylsilyl 69 or t-butyldimethyl- silyl 7o derivatives whereas 1,2-diols and 1,3-diols are conveniently analysed as cyclic boronate derivatives, using EI 69 or CI 71 conditions. An alternative method is based on the analysis of the monoacetyl diglycerides that are formed by acetolysis of the phospholipid^.^^

The identification of 9- and 1 0-trichloromethyl-stearic acids as esterifying acids in phospholipids that were isolated during studies of the metabolism of carbon tetrachloride was accomplished by mass-spectrometric examination of transmethylation products." G.c.-m.s. studies of compounds ( 3 ) and (4), as their bis-trimethylsilyl derivatives, have been reported as the basis of a method for the identification of the polar moiety of phospholipids, foliowing their enzymic hydrolysis and N- demethylation. 74

0

( 3 ) ( 4 )

Miscellaneous Natural Phosphates.-Bombardment with fast atoms has been used to identify ecdysone 22-phosphate and 2-deoxyecdysone 22-phosphate, isolated from eggs of the desert Fast-atom bombardment, used in the negative-ion mode, gave molecular ions at m / z 543 and 527 respectively, whereas field desorption and chemical ionization were unable to provide information on the molecular weight. The ions of highest mass that were seen when using chemical ionization by isobutane were reported at m / z 447 and 431.

Bombardment with fast atoms has also provided mass spectra of intact vitamin BI2 and vitamin B12 coenzyme, the molecular weights being 1354 and 1 578 respectively. l5 , 76 Previously, the spectrum of vitamin B 12 that was obtained by laser-assisted field desorption had been regarded as an extreme achievement in this field.77 6 9 S. J. Gaskell and C. J . W. Brooks, J. Chromatogr., 1977, 142, 469. 70

71

I2

73

14

I5

16

77

K. Satouchi and K. Saito, Biomed. MQSS Spectrom., 1979, 6 , 396. S. J . Gaskell and C. J . W. Brooks, Org. MQSS Spectrom., 1977, 12 , 651. Y. Ohno, I . Yauo, and M. Masui, Adv. MassSpectrom. Biochem. Med., 1977, 2, 559. J . T. Trudell, B. Boesterling, and A. J . Trevor, Roc. Natl. Acad. Sci. USA, 1982, 79, 2678. S. G. Karlander, K. A. Karlsson, and I. Pascher, Biochim. Biophys. Acta, 1973, 3 2 6 , 174. R. E. Isaac, M. E. Rose, H. H. Rees, and T. W. Goodwin, J. Chem. SOC., Chem. Commun., 1982,249. M. Barber, R. S. Bordoli, R. D. Sedgwick, and A. N. Tyler, Biomed. MassSpectrom., 1981, 8 , 492. H.-R. Schulten, W. D. Lehmann, and D. Haaks, Org. MassSpectrom., 1978, 13, 361.

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290 Organophosphorus Chemistry

Monosaccharide phosphates have been analysed successfully as their disodium salts, using field d e ~ o r p t i o n , ~ ~ although these samples form tarry residues on the emitter, making their analysis d i f f i ~ u 1 t . l ~ Dimethyl phosphates of monosaccharides have been analysed under electron-impact conditions, as their cyclic butaneboronate esters.79 The relatively intense (M-C4Hg)+ ion that is seen in these spectra may be used as the basis of a method for the isotopic analysis of these compounds. The field-desorption spectra of pyridoxal 5'-phosphate and pyridoxamine 5'-phosphate have also been recorded." Natural phosphates have been used as test materials for other ionization techniques that are intended for labile materials. For example, intact oestriol 3-phosphate disodium salt has been analysed by laser desorption. l 8

4 Synthetic Compounds

In this section, organophosphorus ester pesticides form a distinct sub- section because of their economic importance and the consequently large body of published work devoted t o their analysis. For many of the compound classes that are dealt with in the following sub-sections, the basic rules of electron- impact fragmentation have been described in three early reviews3' "' 82

Detailed discussion in this Report is therefore reserved for compounds of novel structure, reported subsequently (post 1973). Organophosphorus Ester Pesticides.-Whereas the development of mass spectrometry in the fields of nucleotides and phospholipids has been directed mainly towards the goal of structural analysis, mass spectrometry in pesticide analysis has the further aim of detection of traces of the compounds. Thus, in addition t o spectra giving structural information, there is a requirement for spectra that consist only of one or two diagnostic ions, of high intensity, that are produced under circumstances giving the best possible signal- to-noise ratio. A survey of mass spectrometry in the field of organophosphorus pesticides provides some interesting observations on the tailoring of chemical- ionization techniques t o meet requirements through the choice of the reagent gas.

A thorough review of the mass spectrometry of orgaiiophosphorus esters and their alteration products, covering the literature up t o the end of 1973, and including extensive spectral data and references, was provided by De~marchel ier .~ Some applications of g.c.-m.s. techniques in pesticide analysis have been discussed by VanderVelde and Ryan.83

A first comparison of the electron-impact and positive-ion chemical- ionization spectra of fifteen organophosphorus pesticides and fourteen of their major metabolites was provided by Holmstead et aZ. in 1974.2 Samples 7 R H.-R. Schulten, H. D. Beckey, E. M. Bessel, A. B. Foster, M. Jarman, and J . H .

l9 J . Wiecko and W. R. Sherman, Org. MassSpectrom., 1975, 10, 1007. Westwood, J. Chem. Soc., Chem. Commun., 1973,416.

M. C. Sammons, M. M. Bursey, and D. A. Brent, Biomed. Mass Spectrom., 1974, 1, 169.

" I . Granoth, Top. Phosphorus Chem., 1976, 8 , 41. 8 2 R. G. Gillis and J . L. Occolowitz, Anal. Chem. Phosphonts Compounds, 1972, 295. 83 G. Vaiider Velde and J. F . Ryan, J. Chrornatogr. Sci., 1975, 13, 322.

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Mass Spectrometry o f Organophosphorus Compounds 291

were introduced via the direct-insertion probe. Most of the spectra were recorded while using methane as the reagent gas, so that some fragmentation was evident, although the spectra were generally simpler than those obtained by using EI ionization. The fragmentation that arose when CI was used often differed from that observed on electron-impact ionization, so that EI and CI were regarded as complementary techniques. Ammonia was used as a reagent gas to provide quasi-molecular ions in two cases where methane failed to do

Stan has published a number of papers dealing with the g.c.-m.s. analysis of organophosphorus pesticides, using both CI and EI technique^.^-^^ A survey using three different positive-ion reagent gases, with 23 pesticides, showed that the transition from methane to isobutane to methanol as the reagent gas produced successively simpler spectra, with only (M + H)+ ions being formed when methanol was used. Isobutane, which always produced (M + H)' ions together with some fragmentation, was recommended for routine work." All but two of these 23 compounds could be separated on a capillary column, with detection at the 0.45 p.p.m. level by scanning and down to 40 p.p.b. in a number of cases by monitoring selected ions.87

Monitoring selected ions in the CI mode gave better detection limits than the EI mode.87 Nevertheless, electron-impact ionization formed the basis of a comprehensive classification scheme, based on a repetitive-scanning g.c.-m.s. analysis. Forty-nine compounds could be classified as dimethyl or diethyl phosphorodithioates (5), phosphorothionates (6), phosphorothiolates (7), or phosphates (8) on the basis of the ion intensity at only five values of mass. Typical ions, allowing the separate identification of each compound, are also listed in this paper.s4

so.

S ti

( R'O) ,P-o-R~

0 0 I 1

( R'o),P-s-R~ II (R'o),P-o-R~ U (EtO),P-S-CH,jS-C,H,Cl

(7)

A comparison of five ionization methods, using sixteen organophosphorus pesticides, was carried out by Hass and co-workers in 1 978.88 The ionization methods that were used were electron-impact, positive-ion chemical ioniza- tion (using methane), and negative- ion chemical ionization [using methane

R 4 H. J . Stan, B. Abraham, J . Jung, M. Kellert, and K. Steinland, Fresenius' 2. Anal.

" H. J. Stan, Fresenius'Z. Anal. Chem., 1977, 287, 104. 86 H. J . Stan, Chromatographia, 1977, 10, 233. a 7 H. J. Stan, Z. Lebensm.-Unters. Forsch., 1977, 164, 153.

Chem., 1977,287, 271.

K. L. Busch, M . M . Bursey, J . R. Hass, and G. W. Sovocool, Appl. Spectrosc., 1978, 3 2 , 388.

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292 Organophosphorus Chemistry

(electron-capture spectra), a methane-oxygen mixture, and oxygen as the reagent gases]. The negative-ion spectra, which were characterized by intense phosphate [e.g. ( S a ) ] or aromatic anions [e.g. (9a) , when the latter were suitably substituted] , afforded much higher sensitivity than EI or positive-ion CI. Thus, methane or methane-oxygen negative-ion CI allowed the detection of as little as 75 femtomoles of some compounds by monitoring selected ions.

The use of C1- as the reagent ion with seventeen phosphorodithioate ester pesticides and a single phosphorothiolate ester (demeton) gave simple, characteristic spectra in every case." The spectra again generally showed the anion (R0)2PS2- [(R0)2POS- for demeton] as the base peak, and a chloride- attachment ion (M + Cl)-, giving the molecular weight, was invariably present. Other intense ions were formed when potentially stable anions, e.g. (9a) , were present in the molecule. The chloride-attachment ion allows unequivocal identification in a simple screening method. The temperature dependence of these spectra was also studied.

Field desorption has recently been introduced as a method for the quantitative determination of all four groups (5)-(8) of organophosphorus ester pesticides in waste water.89 The FD spectra show abundant molecular ions and characteristic fragment ions, formed by cleavage a! [for groups (6) and (8)] or p [for groups (5) and ( 7 ) ] to the phosphorus atom. Organo- phosphorus ester pesticides can be identified in waste water at the nanogram level, without prior purification, by the use of high-resolution field deso rp t ion.

Hass and co-workers have recently also reported a preliminary examination of methods of analysis of organophosphorus pesticides, using on-line 1.c.- m.s. techniques2' A system for directly introducing liquids was used for samples that had been separated on a reversed-phase column, using 60/40 acetonitrile/water as the mobile phase. The spectra were reported to be simple and similar to these recorded for methane negative-ion CIYg8 although molecular anions were generally not observed. The method was proposed as being suitable for residue analysis. Phosphates.-The fragmentation of phosphate esters under electron-impact conditions has been reviewed by Granoth8' and by Gillis and Qccolowitz.82 Alkyl and aryl phosphates show both simple bond cleavages and rearrange- ment processes, the latter being more common in the aryl series. Parallel to their greater thermal stability, aryl phosphates are more stable under electron impact, giving much more intense molecular ions. Fragment ions that contain phosphorus are relatively rare in the spectra of aryl phosphates.

Chemical-ionization studies of trialkyl and triaryl phosphates and related phosphoramidates (10) and (1 1) used ammonia as the reagent gas.g0 Unlike

0 . I I

( P r10 ) 2 ~ ~ ~ 2

R9 H.-R. Schulten and S. E. Sun, Int. J . Environ. Anal. Chem., 1981, 10, 247. 90 P. A. Cload and D. W. Hutchinson, Org. Mass Spectrom., 1983, 18, 57 .

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Mass Spec t ro m e try of Organ o p h osp h orus Co m po u n ds 293

the EI spectra, which often showed very weak molecular ions, these CI spectra all showed intense (M +H)+ ions. Fragment ions which gave informa- tion as to the nature and number of groups bonded t o phosphorus were usually present in the CI spectra, although trimethyl phosphate and triphenyl phosphate showed very little fragmentation. Capillary gas chromatography- ammonia chemical ionization was used to identify tri-p-cresyl phosphate (amongst other additives) in ester oils.”

Negative-ion chemical-ionization screening techniques were used by Dougherty 92 to detect tris-( 1,3-dichlor0-2-propyl) phosphate, a flame retardant that has mutagenic properties, in human seminal plasma. Although the screening technique gives only a molecular ion cluster, the elemental composition of the molecular ion could be established (by accurate measure- ment of its mass and a consideration of the pattern of chlorine isotopes) as C4HI504PCl6. Negative-ion spectra of phosphate esters and sulphur analogues that were recorded under electron-impact conditions (i.e. low pressure in the ion source) show characteristic fragment ions, but no molecular ion, unless an additional electron-capturing group is present.93

A number of authors have discussed the electron-impact spectra of the cyclic phosphate esters ( 1 2; X = 0)-( 16; X = 0) and their sulphur [ ( 12; X = S ) and (14; X = S ) ] and selenium analogues (14; X=Se),94-97 in addition t o

X

( 1 2 ) Y = OMe, OPh, OH, 4 - M e 0 - C 6 H 4 0 , SMe, Me, or Phg7

( 1 3 ) R = H or Meg7

( 1 4 ) Y = O H , OMe, SMe,

C1, or SHg4

Me

(15 ig6

” A. Zernan, Fresenius’ 2. Anal. Chem., 1982, 310, 2 4 3 . ’’ T. Hudec, J. Thean, D. Kuehl, and R. C. Dougherty, Science, 1981, 211, 951. 93 H. J . Meyer, F. C. V. Larsson, S. D. Lawesson, and J . H. Bowie, Bull. SOC. Chim.

94 H. Keck, W. Kuchen, and H. F. Mahler, Org. Mass Spectrom., 1980, 15, 59 1. 9 5 K. J . Voorhees, F. D. Hileman, and D. L. Smith, Org. MassSpectrom., 1979, 14, 459. ’‘ A. Murai and M. Kainosho, Org. Mass Spectrom., 1976, 11, 175.

Belg., 1978, 87, 517.

N. Fukuhara and M. Eto, Nippon Noyaku Gakkaishi, 1980, 5 , 6 3 .

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294 Organ o p h osp h or us Ch e m is t ry

X

R bo

( 1 7 ) X = 0, S , o r S e ; Y = NR1R2 x = N R ~ R ' ; Y = 0 , S , o r S e

R1, R2= H , M e , P h , e t c . ( 1 6 ) R = H , M e , E t , o r CH20Hg5

detailed studies of the spectra of diastereoisomeric pairs of the dioxaphos- phorinans (1 7).98-'00

In both monocyclic esters (1 3) and (1 5), migration of hydrogen to oxygen is a first step in the formation of many ions. Thus compound (15), for example, gives an intense ion with the composition [(H0)3P(OC6H5)]'. Rearrangement also takes place with compounds of structure (14). Observa- tion of the direct formation of ions with the composition (C6H4)2X from the parent ion indicates migration of X from phosphorus t o carbon on electron- impact ionization. Migration of other groups, such as methyl, phenyl, and halogen, is not uncommon in the field of phosphorus compounds, making the interpretation of spectra in structural terms difficult in some cases.

Simple cleavage of the P-Y bond is not observed in (14) whereas it is a common mode of fragmentation in many similar compounds. Thus compound (1 2; X = 0, Y = SMe) fragments t o give a ( h l - SMe)' ion. The bicyclic esters ( 16) fragment by loss of e t h ~ l e n e , ' ~ unlike the corresponding phosphites, which lose the elements of formaldehyde (see p. 298).

The EI spectra of enolic phosphate esters (18) show fragmentation patterns that depend on the substitution of the double bond."" lo2 Thus, if R3 = H , loss of an olefin is the major pathway, but when R3 = Me, rearrange-

1 0

( E t 0 ) 2 P O C = C , II ,R

I R R3

H

/ 'OAr H

X 0, ii

MeLI-NRIRz 0 COMe R1O,Il / R O 2 /poc\H

M e Me

(19) ( 2 0 ) X = 0 o r S

W. J . Stec, B. Zielinska, and B. van de Graaf, Org. MassSpectrom., 1980, 1 5 , 105. 99 Z. J. Lesnikowski, W. J . Stec, and B. Zielinska, Org. Mass Spectrom., 1980, 15, 454.

loo B. Zielinska and W. J . Stec, Org. Mass Spectrom., 1978, 13, 6 5 . lo ' E. M. Gaydou and G. Peiffer, Org. Mass Spectrom., 1974, 9, 514. lo* E. M. Gaydou, G. Peiffer, and M . Etienne, Org. Mass Spectrom., 1974, 9, 157.

98

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Mass Spectrometry of Organophosphorus Compounds

C l

295

ment processes involving the enolic chain occur. Phosphoacetoin and its methyl esters (1 9) show mass spectra that are complex mixtures of thermal and electron-impact-induced p r o c e s ~ e s . ' ~ ~

The spectra of some nitrogen-containing analogues of cyclic phosphate esters (20)-(23) have been r e p ~ r t e d . ' ~ - ' ~ ' The stable benzodiazaphosphole unit forms the basis of abundant ions in the spectra of compounds (21). Corresponding fragments are found in the spectrum of compound (22). Both of these structures also fragment by cleavage of both P-N bond^."^' lo6 The chlorine-containing compounds of structure (23) fragment initially by a retro-Diels-Alder reaction to give ( M - P02C1)+ ions, which then undergo further fragmentation.lW The presence of amino-substituents on the phosphorus atom of compounds of structure (20) results in preferential cleavage in this substituent and cleavage of the P-N bond.'07 Thus ions at m / z 167 (C5H12P02S)+ and 133 (C5H10P02)+ are characteristic of the sulphides (20; X = S).

Cyclophosphamide (24) and a number of its metabolites which are not amenable to electron-impact analysis may be identified by field desorption.lo8 Field desorption has also been used for the quantitative determination of cyclophosphamide and its metabolites. ' 09 , Another report describes electron-impact ionization (following methylation) for the same analysis."'

The activated intermediates that are derived from cyclophosphamide are hydroperoxides. Even under field-desorption conditions, a molecular ion is difficult to detect for these compounds, and (M - H2O)' is the most signifi- cant ion in this region. The quantitative analysis of these intermediates may be accomplished by allowing them to react with benzyl mercaptan, which provides benzyl thioethers; these give excellent EI spectra. '12

The products arising from thermal decomposition of the 00 '-diethy1

S. Meyerson, E. S. Kuhn, F. Ramirez, J . F. Marecek, and H. Okazaki, J. Am. Chem. SOC., 1980, 102, 2398.

I o 4 V. N . Gogte, P. S. Kulkarni, A. S. Modak, and B. D. Tilak, Org. Mass Spectrom., 1981, 16, 5 1 5 .

lo' M. S. R. Naidu and C. D. Reddy, Indian J. Chem., Sect. B, 1977, 15, 706. lo6 M. S. R. Naidu, C. D. Reddy, and P. S. Reddy, Indian J. Chem., Sect. B, 1979, 17,

I o 7 R. S. Edmundson, Phosphorus Sulfur, 1981, 9, 307. I o 8 H.- R. Schulten, Biomed. Mass Spectrom., 1974, 1, 22 3. log H. D. Lehmann and H.-R. Schulten, 2. Anal. Chem., 1978, 290, 121. ' l o U . Bahr, and H.-R. Schulten, Biomed. MassSpectrom., 1981, 8 , 5 5 3 .

458.

R. F. Struck, M. C. Kirk, M . H. Witt, and W. R. Laster, Jr., Biomed. Mass Spectrom., 1975, 2 , 46.

1 1 2 M. Przybylski, H. Ringsdorf, U. Lenssen, G. Peter, G. Voelcker, T. Wagner, and H. J . Hohorst, Biomed. Mass Spectrom., 1977, 4, 209.

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296 Organophosphorus Chemistry

S-butyl ester of dithiophosphoric acid and of 00 '-diethy1 phosphorothionyl disulphide have been studied, using g.c.-m.s. techniques. '13

Phosphonates.-A detailed study, based on the EI spectra of 24 alkyl and six aryl phosphonates, has been described by Occolowitz and Swan114 and the spectra of phosphonate esters have subsequently been r e ~ i e w e d . ~ , ' 22 Many saturated dialkyl phosphonates (25) show the same base peak [i.e. HP(OH)3+] as trialkyl phosphites whereas the ion R'P(OH)3' is usually the base peak in the EI spectra of dialkyl alkylphosphonates (26), which are isomeric with the corresponding trialkyl phosphites.

0 II

R I P ( OR^ ) ( 2 6 ) ( 2 7 ) X = C H 2 , C H C 1 , C H B r , o r C B r 2

0 II

( E t 0 ) 2 P C H R X

( 2 8 ) R = H; X = C O O E t , C N , CH=CH2, o r COOH R = M e ; X = COOEt

0 0

( E t 0 ) 2 P - C O C H 2 P h II ll

Whilst the molecular ion in the EI spectra of phosphonates is frequently weak, the CI spectra that were obtained by using both ammonia and hydro- carbon as the reagent gas '15 gave intense (M+H)' ions. Useful structural data could also be obtained from the ammonia CI spectra, which included those of a number of bisphosphonate esters (27) and of variously substituted phosphonate esters (28) and (29).'*

0 0 0 I1 I

Me2N-P-OEt II

~e - P - s R~ I

II I

Me-P-OR

F CN OR2

A comparison of chemical ionization and electron-impact ionization of a range of alkyl phosphonates, phosphonofluoridates (30), phosphonothiolates (3 l) , and an amidophosphorocyanidate (32) favoured chemical ionization as an identification t e~hn ique . "~ Methane reagent gas gave spectra with quasi- molecular and fragment ions whereas ethylene and isobutane gave simpler spectra, with more intense quasi-molecular ions. The best sensitivity was achieved by using ethylene both as the carrier gas in the gas chromatograph and as the reagent gas. Negative-ion spectra of phosphonate esters and of sulphur analogues, recorded under electron-impact conditions, show

V. Trdlicka, J . Mitera, and J . Mostecky, BrdoeIKohIe, Erdgas, Petrochem., 1977, 30, 332.

'I4 J. L. Occolowitz and J . M. Swan, Aust. J. Chem., 1966, 19, 1187. 'I5 S. Sass and T. L. Fisher, Ox. Mass Spectrom., 1979, 14, 2 5 7 .

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Mass Spectrometry of Organophosphorus Compounds 297

characteristic fragment ions, but no molecular ion unless an additional electron-capturing group is p re~en t . ' ~

A number of trichloromethyl-substituted cyclic esters and nitrogen analogues (33)-(35), containing five- and six-membered rings, have been prepared and their EI spectra examined.l16 These authors compared the fragmentation of the dioxaphospholans (33) (which produce four-membered cyclic ions by ring-contraction) and of the dioxaphosphorinans (34) with that of compounds that contain P-N [e.g. (35)] and P-S bonds, which mostly undergo cleavage of the P-C bond to give the base peak. In another recent study,"' the electron-impact spectra of a series of linear phosphonoacetals (36) were compared with those of the cyclic compounds (37). Spectra have been reported for the aromatic system (38) ' 1 8 and for acetylenic dialkyl- and y6 -ethylenic 0-keto-phosphonates. '19

(36) n = 1 or 3

0 /O\CHCHZP( II OEt ) 2

I /-\

S Y ( H2C ) n J O

(38) Y = OH, OEt, or C1 ( 3 7 ) n = 0 or 1

The fragmentation (under electron impact) of the acid chloride (39; R = Cl), which is used in the synthesis of compounds (33)-(35) and of other acid chlorides with the general formula (39), had also been documented in an earlier paper.'20 The compounds (39) give carbonyl fragment ions (RCO)' by C1-0 exchange. The EI spectra of the acid halides (40) and (41) have been recorded by Miller and co-workers.121

0 S 0 II II

C6F5P(Hal)2 C6F5PF2 I1

RCCl 2PC 1

(39) ( 4 0 ) ( 4 1 )

B. M. Kwon and D. Y. Oh, PhosphomsSulfur, 1981, 11, 177. ' I ' S. Yanai, PhosphorusSulfur, 1982, 12, 369. "* M. S. Bhatia and Pawanjit, Org. M~ssSpec t rom. , 1977, 12, 1.

"' T. R. B. Jones, J . M. Miller, and M. Fild, Org. Mass Spectrom., 1977, 12, 317.

G. Peiffer and E. M. Gaydou, Org. MQSS Spectrom., 1975, 10, 122. G. Schmidtberg, G. Haegele, and G. Bauer, Org. MQSS Spectrom., 1974, 9, 844.

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298 Organ o p h 0 s p h orus Ch e m is try

Phosphonoacetic acid has been determined quantitatively as its tris- trimethylsilyl ether, using methane chemical ionization to give a spectrum comprising only (M + H)+ and (M - CH3)+ ions.'22 Trimethylsilyl derivatives of alkyl- and aminoalkyl- phosphonic acids 123 and N- trifluoroacetyl n-butyl esters of (aminoalky1)phosphonic acids 124 have also been prepared for g.c.- m.s. analysis. Field desorption has been used to determine the molecular weights of a series of phosphonate- and phosphate-ester-based macrocyclic compounds.'25 P1iosphites.-The EI spectra of aryl and alkyl esters have been reviewed.'" 82

Alkyl phosphites generally give very weak molecular ions and a base peak which contains phosphorus, oxygen, and hydrogen only, e.g. HP(OH)3+. The base peak in the spectrum of triphenyl phosphite ( m / z 94) has the composition of phenol.

R

( 4 2 ) R = H , Me, E t , o r C H 2 0 H

Several authors 957 1269 12' have studied the EI mass spectra of bicyclic esters of formula (42), particularly since some compounds of this structure are extremely toxic for mammals. A specific loss of formaldehyde is suffi- cient t o distinguish these compounds from the corresponding phos- p h a t e ~ . ~ ~ , 126 Mass spectrometry has been used for the identification of phosphite and thiophosphite esters. 12'

Ammonia chemical-ionization studies of trialkyl and triphenyl phosphites show ( M + H ) + ions, carrying at least 10% of the total ion current.g0 Some fragmentation is observed. For example, the major fragmentation for triphenyl phosphite was the loss of phenol from (M + H)+. Negative-ion spectra of phosphite esters that were recorded at low electron energy ( G 8 eV) showed mainly (M - R)- ions, formed by a dissociative electron-capture process. 129

Phosphinates.-A substantial body of work on the electron-impact spectra of phosphinic acids and phosphinate esters has been reviewed by Granoth '' and De~marchel ier ,~ and particularly thoroughly by Gillis and Occolowitz.'* Since then, the electron-impact spectra of steroid esters of dimethylphosphinic acid

J . R o b o z , R. Suzuki, G. Bekesi, and R. Hunt, Biomed. Mass Spectrom., 1977, 4, 29 1 . D. J . Harvey and M . G. Homing, Org. Mass Spectrom., 1979, 9, 1 1 1 .

V. G. Golovatyi and E. N. Korol, Teor. Eksp. Khim., 1981, 17, 849. H. Kenttamaa and J . Enquist, Org. Mass Spectrom., 1980, 15 , 5 2 0 .

F. Bartonicek, J . Hrusovsky, and L. Hanus, Vet. Med. (Prague), 1977, 22, 713. I . I . Furlei, U. M . Dzhemilev, V. I . Khvostenko, and G. A. Tolstikov, Izu. Akad. Nauk SSSR, Ser. Khim., 1976, 2 127.

124 M. L. Rueppel, L. A. Suba, and J . T. Marvel, Biomed. MassSpectrom., 1976, 3, 28. 1 2 5

' 2 7 D. G. Hendricker, J. Heterocycl. Chem., 1967, 4, 385.

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Mass Spectrometry o f Organophosphorus Compounds 299

and of dimethylthiophosphinic acid (43),130 perfluoroalkylphosphinate esters (44; R1 = per f lu~roa lky l ) , ' ~~ and two substituted cyclic acids (45) 13* have appeared in the literature. The spectra of phosphinic acid halides with the general formula (46) 1219133 and of the azides (47) 133 have also been published.

X II

Me 2P-O- s t e roi d

(43) X = 0 or S

X

1 2 1 1 R R P H a l

0 II

E t ( R1 )POR2

(44) Y

(45) X = Br, Y = H

X = H, Y = Br

( 4 6 ) X = 0 , S , or Se (47) X = 0, S , or Se

Phosphines.-A number of detailed studies of electron-impact spectra of phosphines have been described, following earlier reviews of this subject.81 ' 82

These studies covered triphenyl-, tritolyl-, and tri-(2,6-dimethylphenyl)-,lX d i ~ h e n y l - , ' ~ ~ and mixed aryl(alky1)- p h o ~ p h i n e s , ' ~ ~ ' 137 aminomethyl-, 1373

h y d r ~ x y m e t h y l - , ' ~ ~ cyano- (48),'39 and acyl-substituted phosphines (49),'37 tri-( p -alkylphenyl)phosphines,lm and neopentyl- substituted phosphines. 14' When a comparison was made of the ease of cleavage of phosphorus-aryl

R2P( CH=CH)n CN X C O P R ~ R~ R 1 2 P ( CH2)n PR2R3

( 4 8 ) n = 0 or 1 ( 4 9 ) X = a l k y l , OMe, (50 ) R1= a l k y l or a r y l

R2= a l k y l or H R3= alkyl or a r y l

Me2N, or NHPh

I3O K. Jacob, W. Vogt. M. Knedel, and W. Schaefer, Biomed. Mass Spectrom., 1976, 3 ,

1 3 1 A. V. Garabadzhiu, A. A. Kodin, A. N. Lavrent'ev, and E. G. Sochilin, Zh. Obshch. 64.

Khim., 1981, 51, 41. K. Moedritzer and R. E. Miller, PhosphorusSulfur, 1981, 10, 279.

1 3 3 H. F. Schroeder and J. Mueller, 2. Anorg. Allg. Chem., 1979, 451, 158. 134 T. R. Spalding, Org. Mass Spectrom., 1976, 11, 1019. 13' K. Henrick, M. Mickiewicz, and S. B. Wild, Aust. J . Chem., 1975, 28, 1455. 136 K. Henrick, M. Mickiewicz, N. Roberts, E. Shewchuk, and S . B. Wild, Aust. J. Chem.,

1975,28, 1473. R. G. Kostyanovsky, A. P. Pleshkova, V. N. Voznesensky, and Yu. I. El'Natanov, Org. Mass Spectrom., 1976, 11, 237.

13' R. G. Kostyanovsky, V. N. Voznesensky, G. K. Kadorkha, and Yu. I. El'Natanov, 0%. Mass Spectrom., 1980, 15. 412.

13' R. G. Kostyanovsky, A. P. Pleshkova, V. N. Voznesensky, and Yu. 1. EI'Natanov, Org. MassSpectrom., 1980, 15, 397. G. Marshall, S. Franks, and F. R. Hartley, Org. MassSpectrom., 1981, 16, 272.

14' R. B. King, J. C. Cloyd, Jr., and R. H. Reimann, Org. MassSpectrom., 1976, 11, 148.

137

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300 Organophosphorus Chemistry

bonds in mixed aryl-phosphines, phenyl was found to be more readily lost than p-anisyl, which in turn was more readily lost than p-dimethylamino- phenyl. 142 Dissociative electron-capture spectra of phosphines R3P, which comprise mainly (M - R)- ions, were recorded in another publication.128 The spectra of phosphines (50) that contain more than one phosphorus atom have also been reported in two papers. 14' , 143

Ar R

Ph2 PCH= CHEPh2

( 5 5 ) E = P o r A s f7JJ-J R

EPh2 Ph2P( CH2 )2AsPh2

( 5 6 )

aPPh2 \ / I

( 5 3 )146 ( 5 4 ) E = P , A s , or Sb

A number of heterocyclic phosphines ( 5 1)- - (53) have been subjected to analysis under electron-impact conditions 144-146 and the spectra of phosphine-related o -phenylene chelating agents (54) that contain phosphorus, arsenic, or antimony donor atoms have been discussed by Sedgwick and co- w o r k e r ~ . ~ ~ ' The EI spectra of other diphosphine and phosphine-arsine ligands (55) and (56) have also been p ~ b 1 i s h e d . l ~ ~ ' 149

A review of the spectra of phosphine oxides and sulphides" has been supplemented by EI data on tri-( p-alkylpheny1)phosphine oxides,'40 diphenylphosphine s ~ l p h i d e s , ' ~ ~ neopentylphosphine sulphides and and the cyclic phosphine oxides (57; X = O ) and sulphides (57; X = S ) and (58).15'

14' L. Horner and U. M. Duda, Phosphorus, 1975, 5 , 135. 143 J. C. Briggs, C. A. McAuliffe, W. E. Hill, D. M. A. Minahan, and G. Dyer, J. Chem.

144 W. D. Weringa and I. Granoth, Org. Mass Spectrom., 1973, 7 , 459. SOC., Perkin Trans. 2, 1982, 321.

T. R. B. Jones, J. M. Miller, S. A. Gardner, and M. D. Rausch, Can. J. Chem., 1979,

146 V. N. Bochkarev, A. N. Polivanov, V. I. Aksenov, E. F. Bugarenko, and E. A. Cherny-

147 W. J. Kevason, C. A. McAuliffe, and R. D. Sedgwick, J. Organomet. Chem., 1975, 84,

14' K. K. Chow and C. A. McAuliffe, J. Organomet. Chem., 1973, 59 , 247. 149 G. Cauquis, B. Divisia, and J. Ulrich, Org. MassSpectrom., 1975, 10, 1021.

l S 1 M. G, Voronkov, V. Yu. Vitkovskii, N. W. Kudyakav, and R. K. Valetdinov, Zh.

145

57, 335.

shev, Zh. Obshch. Khim., 1974, 44, 1273.

239.

G. Cauquis, B. Divisia, and J. Ulrich, Org. Mass Spectrom., 1975, 10, 770.

Obshch. Khim., 1981, 5 1 , 2176.

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Mass Spectrometry of Organophosphorus Compounds 301

/ \ X R

( 5 7 ) 146 ( 5 9 ) X = 0 or S

A comparison of the spectra of a number of cyclic phosphine oxides and sulphides with the general formula (59) showed that there are more intense molecular ions when X = S and that there is a reduction in the effect of the keto-group on fragmentation in the spectra of the ~ u l p h i d e s . ' ~ ~ Positive-ion chemical ionization, using methane or isobutane, afforded intense (M + H)' ions from a number of alkyldiphenylphosphine oxides which only gave weak M+ and (M + H)' ions under electron-impact condition^.'^^

s s R2P-PR2

I1 II s s

R( M e )P-P( M e ) R I1 II

i i Ph2P-X-PPh2

P Ph2PR

( 6 3 ) X = C H 2 , CH=CH, o r CN2 ( 6 4 )

A study of the spectra of a number of diphosphine disulphides (60) and (61) showed that, unlike the case of the sulphides of trisubstituted phos- phines,81 a direct loss of sulphur from the molecular ion is not common.154 A rearrangement t o a four-membered ring (62), prior t o fragmentation, was suggested for these compounds. Disulphides with the general formula (63) gave very complex spectra, with evidence for migration of phenyl groups from one phosphorus atom to another, or to sulphur, or to electrophilic carbon atoms.'49 Migration of sulphur atoms was also observed. Migration of phenyl or methyl groups occurs in the related monosulphides (64).'"

Ph3RP+ H a l - + +

Ph3P(CH2)nPPh3 Hal-

Phosphonium Salts.-The direct analysis of phosphonium salts only became possible with the introduction of field-desorption techniques. Field-desorp- tion analysis of six monophosphonium halides (65) showed the cation as the base peak, although the anion was normally difficult t o determine by this '" A. E. Lyuts, V. V. Zamkova, A. P. Logunov, Z . A . Abrarnova, B. M. Butin, and Yu.

I s 3 S. D. Goff, B. L. Jelus, and E. E. Schweizer, Org. Mass Spectrom., 1977, 12, 33. G. Bosyakov, Izv . Akad. Nauk Kaz . SSR, Ser. Khim., 1979, No. 2, p. 20.

H. Keck and W. Kuchen, Org. Mass Spectrom., 1979, 14, 149.

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302 Organophosphorus Chemistry

t e c h n i q ~ e . ' ~ ' In the spectra of a number of bisphosphonium halides (66), the base peak represented two cations, associated with an anion."'

Phosphonium salts with the general formula (67) underwent cleavage of the P-aryl bond on field ionization t o give phosphine cation radi~a1s. l~ ' The thermolysis of (1 -methoxycarbonylalkyl)triphenylphosphonium bromides has been studied, using electron-impact i o n i ~ a t i o n . ' ~ ~

Ph3P=C /R1

P h a A r l b A r 2 P+ B r - \R2

( 6 7 ) a + b + c = 4 ( 6 8 ) ( 6 9 )

Stabilized phosphonium salts with the general formula ( 6 8 ) , which were originally extensively studied by Cooks et were also the subject of a later paper.lS8 In this case,158 the phosphinyl-stabilized compounds (69) gave strong M+ and (M - H)+ ions, together with fragment ions that are representa- tive of the phosphonate and phosphonium moieties under electron impact. Phosphazenes.-Hexakis(polyfluoroalkoxy)cyclotriphosphazenes (70) have been synthesized and thoroughly investigated as reference compounds, of high molecular weight, for mass s p e ~ t r o m e t r y . ' ' ~ These compounds, which are relatively volatile and easily synthesized, are particularly suitable for work with soft ionization techniques, in the mass range 1000-2000. Phosphonitrile chlorides (PNC12), have been suggested as reference compounds for negative- ion chemical-ionization operation. 160

Me

X / N \

\N/PF2Y

R

( 7 1 ) X = C=O o r PF Y 2 ( 7 0 )

( 7 2 )

l S s G. W. Wood, J. M. Mclntosh, and P.-Y. Lau, J. Org. Chem., 1975, 40, 636. 1 5 6 F. Sanchez-Ferrando and A. Virgili, An. Quim., 1977, 73, 1059. I s 7 R. G. Cooks, R. S. Ward, D. H. Williams, M. A. Shaw, and J. C. Tebby, Tetrahedron,

l s 8 L. Toekes and G. H. Jones, Org. M~ssSpectrom., 1975, 10, 241. 1 5 9 K. L. Olsen, K. L. Rinehart , Jr., a n d J. Carter Cook, Jr., Horned. Muss Spectrom.,

I6O Y. Hirata, K. Matsurnoto, and T. Takeuchi , Org. Muss Spectrom., 1978, 13, 11 1.

1968 ,24 , 3289.

1977, 4, 284.

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Mass Spectrometry of Organophosphorus Compounds 303

The spectra of the phosphadiazetidines (7 1) have been discussed in two publications.'61y The disphosphadiazetidines (71; X = PF2Y) show an electron-impact-induced fragmentation t o give M / 2 + 1 , M/2 , and M/2 - 1 as the most abundant ions.'61 The spectrum of the cyclophosphazene (72), which shows anti-tumour activity, has recently been p ~ b 1 i s h e d . l ~ ~ Miscellaneous.-Whilst the present Report in no way claims t o be exhaustive, some other organophosphorus compounds that are not classified within the preceding sections have been noted in the literature of mass spectrometry, viz. a cubane-like P-N compound (73),'@ tin-containing phosphate esters (74),16' (75),'66 phosphorus-containing hydrazides (76) and (77),'67 a series of phosphorus-containing carbamates (78),16' (79),'69 di- and tri-peptide analogues that contain aminomethylphosphonic acid groups (80),' organic hypophosphates and their mono- and dithio-derivatives,"' some simple phosphor in^,'^' and closo-phosphorimide and closo - t h i o p h o ~ p h o r i m i d e s . ' ~ ~

0 I II II

[ MeN-PF2 ] ( MeNCNMe )

0

R3SnOP( O P h ) 2 II

( 7 3 ) ( 7 4 ) ( 7 5 ) X = 0 o r S

0 0 0

I I (ArO)2PNHNH2 II R 1211 R PNPhCOR' Ph2PNHNH2

The thermal depolymerization of polymers with the general formula (8 1) has been studied by heating them whilst they are on the sample probe of the mass ~ p e c t r o m e t e r . ' ~ ~ Polymer (8 la) decomposed t o form cyclic oligomers,

l b l 0. Schlak, R. Schmutzler , and I . K. Gregor, Org. MassSpectrom., 1974, 9, 582. 16' M. A. Baldwin, A. G. Loudon , R. E. Dummer, R. Schmutzler , and I . K. Gregor, Org.

1 6 3 B. Monsarrat , J.-C. Prome, J.-F. Labarre, F. Sournies, and J . C. Van d e Grampel , Mass Spectrom., 1977, 12, 275.

Biomed. Mass Spectrom., 1980, 7 , 405 . K. Utvary, M . Kubjacek, a n d K. Varmuza, 2. Anorg. Allg. Chem., 1979, 458 , 281 . K. G. Molloy, F. A. K. Nasser, a n d J . J. Zuckerman, Inorg. Chem., 1982, 21, 1711. S. W. Ng and J. J . Zuckerman, Organometallics, 1982, 1, 714.

1 6 7 M. E l -Deek , J . Chem. Eng. Data, 1980, 25 , 171. 16' V. A. Kolesova and Yu. A. Strepikheev, Zh. Obshch. Khim., 1979, 49, 2213 . 1 6 9 V. A. Kolesova, Yu. A. Strepikheev, and V. A. Valavoi, Tr.-Mosk. Khim: Tekhnol.

17' K. Yamauchi, Y. Mitsuda, and M. Kinoshita, Org. MassSpectrom., 1977, 12, 119. 1 7 ' W. J. S t ec , B. Zielinska, and J. R. Van Wazer, Org. MassSpectrom., 1975, 10, 485.

C. Jongsma and F. Bickelhaupt, Org. Mass Spectrom., 1975, 10, 515. 17' A. Wolff, A. Cambon , and J. Riess, Org. Mass Spectrom., 1974, 9, 594.

A. Ballistreri, S. Foti , G. Montaudo, S. Lora and G. Pezzin, Makromol. Chem., 1981, 182. 1319.

Inst. im. D. I . Mendeleeva, 1977, 94 , 38.

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304 Organophosphorus Chemistry

( 8 1 ) a ; R = O n a p h t h y l

b ; R = N H A r

c ; R = 1 - p i p e r i d y l

whereas polymers of the type (81b) decomposed with the evolution of amines. Polymer (8 1 c) decomposed completely, t o give ammonia and elemental phosphorus.

The isotopic analysis of organophosphorus compounds has been described in two papers.'75' 176

W. Reimschussel and P. Paneth, Org. Mass Spectrom., 1980, 15 , 302. V. I . Mosichev and 8. B. Alipov, Khim. Tekhnol. Irot. Mechenykh Soedin., 1977, N o . 1, p. 19.

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