Electron Capture Gas Chromatography and Mass Spectral Studies of lodomethyltetramethylmethyldisiloxane Esters and lodomethyldimethylsilyl Ethers of Some Short-Chain Acids, Hydroxy Acids, and Alcohols John B. Brooks, John A. Llddle, and Cynthia C. Alley Center for Disease Control, Public Health Service, U S . Department of Health, Education, and Welfare, Atlanta, Ga.30333.
A practical procedure was developed for the preparatlon of iodomethyitetramethyimethyidisiioxane (IMTMMDS) esters of carboxylic acids and lodomethyldimethyisiiyi (IMDMS) ethers of alcohols. The mass spectra of the esters and ethers were studied. These studies conflrm that the above type derivatives had been formed from the acids and alcohols. Studies with '*O labeled acetic acid confirmed that most of the oxygen used In the formation of the disiioxane group came from the acid. The derivatives formed have a high affinity for free electrons and provide an excellent derivative for the detection of carboxylic acids and alcohols in spent culture media and in body flulds by electron capture gas chromatography and mass spectrometry.
A practical reproducible derivatization procedure that would produce highly electron capturing derivatives of some of the short-chain carboxylic acids (C-1-C-8) for analysis by electron capture gas-liquid chromatography (EC-GLC) would be of value for the analysis of bacterial metabolites both in body fluids and in spent culture media. A method was proposed (1)which employed the use of bromomethyldimethylchlorosilane (BMDCS) derivatives and met most of the above requirements. In later studies, we found we could decrease the background associated with the products of the derivatization reaction by using less BMDCS reagent and, as reported ( 2 ) , produce a more highly electron capturing derivative by replacing the bromine group after derivatization with an iodine group. T o our knowledge, no one has investigated the reaction mechanism and products formed from the reaction of carboxylic acids with BMDCS and subsequent replacement of bromine with iodine. Neither have the electron capturing properties or mass spectral characteristics of the above derivatives of carboxylic acids, hydroxy acids, or short-chain alcohols been investigated. The EC-GLC and mass spectral characteristics of some of the short-chain hydroxy acids and alcohols would be of interest because they too form derivatives with BMDCS and are frequently detected in the spent culture media of bacteria and in body fluids, along with short-chain carboxylic acids. Several investigators have studied the properties of halomethyldimethylsilyl ethers of steroids (3-5). They report that the derivatives are relatively stable to hydrolysis and more suitable for quantitative work than trimethylsilyl ethers. The iodomethyldimethylsilyl (IMDMS) ethers of steroids were reported to have a high affinity for free electrons, and they can be quantitatively prepared from BMDCS derivatives by incubating the derivative in a saturated solution of sodium iodide in acetone at 37 "C for 30 minutes ( 5 ) . Mass spectral properties of chloromethyldimethylsilyl ethers of steroids have been investigated, and their fragmentation properties have been reported ( 5 ) .The present study was conducted to investigate the EC-GLC 1960
0
and mass spectral characteristics of the BMDCS and IMDCS derivatives of the above compounds and to elucidate the reduction mechanism and end products of the reaction.
EXPERIMENTAL Preparation of Standards. The alcohol and carboxylic acid standards consisting of 1-propanol, 1-butanol, isoamyl alcohol, 1pentanol, 2-hexanol, 1-hexanol, 1-heptanol, and /3-phenylethyl alcohol, formic, acetic, propionic, isobutyric, butyric, isovaleric, valeric, isocaproic, caproic, heptanoic, and octanoic acids were prepared as described ( I ) . Individual standards of lactic and a-hydroxy isovaleric acids were prepared by diluting 3.6 fimoles of each to 10 ml with ethyl ether (Baker's analyzed reagent grade containing ethanol as a stabilizer). An aqueous lactic acid standard was prepared by adding 10.8 X mole of lactic acid to 2 ml of water. Preparation of Derivatives. The 2-ml aqueous standard solutions of lactic acid were placed in 50-ml screwcap centrifuge tubes and acidified to about pH 2 with 0.2 ml of 50% (v/v) The acidified samples were then extracted by vigorous shaking with 20 ml of diethylether. After brief centrifugation, the ether layer (top) was decanted into a 50-ml beaker and evaporated with a gentle stream of clean dry air to about 1 ml. The aqueous bottom layer was discarded. Next the concentrate was transferred to a 12- X 75-mm test tube with a disposable Pasteur pipet. Care was taken to discard any visible layer of moisture (bottom) that sometimes is present in the pipet. Then about 100 mg (-0.1 the volume of the concentrated sample) of MgS04 was added. The contents of the tube were thoroughly mixed by shaking. The sample was briefly centrifuged and the ether layer was decanted into a second test tube. One ml of ether was added to the sedimented MgS04; then the tube was shaken to mix the contents and briefly centrifuged. The ether layer was decanted and combined with the previously decanted ether layer. The MgS04 was discarded. At this point, both the dried extracts and 0.1 ml of the standard solutions in organic solvent were treated alike. They were evaporated in a 12- x 75-mm test tube by clean dry air to almost dryness (1drop or less). Next was added from the tip of a disposable Pasteur pipet (-0.01 ml) 1 drop of a 1 part diethylamine (DEA) to 11 parts chloroform as a catalyst and 1 drop of a 1part BMDCS to 11 parts chloroform. The test tube containing the reaction mixture was shaken to mix the contents. The test tube was corked, taped, and heated in an 80 "C water bath for 1 hour. The sample was then removed from the bath and cooled under tap water, and the chloroform was evaporated by air. Next 0.1 ml of a saturated (25 "C) solution of NaI in acetone was added. The test tube was shaken to mix the contents, corked, taped, and incubated for 30 minutes at 35 "C. The sample was then removed from the incubator, and 0.9 ml of chloroform was added. The sample was corked and briefly centrifuged. Next the supernate was decanted into a second test tube and the chloroform was evaporated by air to almost dryness; then 1 ml of xylene was added and 0.5 wl of the sample was inserted into the gas chromatograph, or the sample was corked and stored for future GLC analysis. Gas Chromatography Analysis. A Perkin-Elmer gas chromatograph Model 900 equipped with a 63Ni 10-mCi frequency pulse modulated detector, Beckman 4-way and 3-way switching valves, and a 10-inch (25.4 cm) potentiometric recorder was used. The instrument was operated with two coiled glass columns (0.3-cm i.d. by 7.3-m length). One column (nonpolar) was packed with Chromosorb W 80/lOO mesh (AW-DMCS H.P.) coated with 3% OV-1 (Applied Science Laboratories). The second column (polar) was packed with TA33 Tabsorb (Regis Chemical Co.). The switching
ANALYTICAL CHEMISTRY, VOL. 47, NO. 12, OCTOBER 1975
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Figure 1. lodomethyltetramethylrnethyldisiloxane esters of an acid standard mixture (chromatograms A and C)and iodomethyldimethylsilyl ethers of an alcohol standard mixture (curve B) (RS) residual solvent: (P) by-products: (1) formic: (2) acetic: (3) propionic: (4) isobutyric; ( 5 ) butyric: (6) isovaleric: (7)valeric: (8) isocaproic; (9) caproic; (10) heptanoic; (1 1) octanoic: (a)ethanol: ( b ) propanol: ( c )butanol: (d)isoamyl: (e) amyl: (02-hexanol: (9)1-hexanol: ( h ) heptanol; (Q P-phenylethyl aicohol: (1) nonyi alcohol
valves permitted a comparative analysis of the derivatives on either of the above columns from a single detector and the venting of the column gas when not in use. The instrument was programmed from 100 "C for the TAB column and from 125 "C for the OV-1 column to 225 "C a t a linear increase of 3 "C per minute; it was programmed to hold a t 225 "C for 32 minutes. The temperature of the injector was 225 "C; the manifold, 250 "C; and the detector, 275 "C. The electrometer was set so that 5.12 X lo4 Hz gave full scale response and the standing current was set a t 2.0 nA. A mixture of argon (95%) and methane (5%) (Matheson) was used as the carrier gas a t a flow rate of 50 ml/minute. The transfer gas line was modified between the manifold and detector to permit a flush gas to be used. Use of a flush gas improved the base line and overload characteristic of the detector and also supplied carrier gas to the detector while the columns were being vented. The flush gas was regulated through the detector so that the combined flow of argonmethane from the column and flushing system was 67 ml/minute. The recorder was operated with an input signal of 1 mV. New columns were conditioned for 12 hours a t 245 "C, and longer if necessary, to obtain a good base line. The columns were also conditioned each morning before use by heating them a t 245 "X:for 30 minutes. Mass Spectra. An LKB Model 9000 gas chromatograph-mass spectrometer was used. The resolution of the instrument was about 1000, the temperature of the ion source was 290, and the electron energy was 70 h.Acceleration voltage was 3.5 kV; the scan (rnle) limits were from 0 to 500 rnle; scan speed was 6 (0 to 500 rnle in 16 seconds); and the UV oscillograph chart speed was 5 cm per second. The gas chromatograph was equipped with a coiled glass column (0.3-cm i.d. by 3.6-m length) packed with 3% OV-1 on Chromosorb W 80/100 mesh (AW-DMCS H.P.). The GC effluent was monitored by a total ion current detector. The gas chromatograph was operated isothermally for 5 minutes a t 70 "C; then it was programmed linearly 5 " C per minute to 225 "C. Helium was used as the carrier gas a t a flow rate of 36 ml per minute. The recorder was operated with an input signal of 2 mV (full scale).
RESULTS AND DISCUSSION As reported ( I ) , the standard mixture of acids was detected as BMDCS derivatives in picomole or low nanomole quantities. Conversion of the brominated derivatives to iodized derivatives resulted in a tenfold or better increase in sensitivity and, in addition, permitted a twofold dilution of the reagents, which results in a much cleaner background. After conversion of the brominated ester or ether to the iodine derivative with NaI in acetone, 0.9 ml of acetone can be added to the reaction mixture (final volume 1 ml), and the derivative may be added directly to the gas chromatograph for analysis; however, addition of chloroform and then centrifugation to remove the resulting precipitate produces a slightly cleaner background. Evaporation of chloroform and addition of xylene as a final solvent ensures a constant volume for the derivative and permits storage of the sample a t room temperature for weeks. Derivatives were formed from both the alcohol and acid standard mixtures that had high affinity for free electrons (Figure 1, chromatograms B and C). The iodized derivatives eluted from the TAB (polar) column (Figure 1, chromatogram A ) much faster than they did from the OV-1 (nonpolar) column (Figure 1, chromatogram C), even though the initial temperature of the polar column was 25 OC lower than that of the nonpolar column. Additionally, the retention times of the acids changed in relation to the peaks labeled P (the by-products of the reactions), which indicates that the by-products have different structures from the acid derivatives. As shown (Figure 2) in the mass spectrum of 2-hexanol (spectrum C), butyric acid (spectrum B ) , and lactic acid (spectrum A ) , two different types of derivatives are formed with the acids and the alcohols. A generalized summary of the major reactions is as follows: 1
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where P* = by-products of the reaction and DEA = diethylamine. Intense peaks a t mle 245 and 273 and the metastable peak a t 219.9 are present in the spectra of all the acids but absent in the spectra of all alcohols in the standard mixture (Figure 1, chromatogram B ) . The mass spectrum of each alcohol contained ion fragments at mle 75, 171, 173, and 199 but these were not present in any of the acid spectra. The mass spectrum of IMDMS derivative of 2-hexanol (Figure 2, spectrum C) is typical of all alcohols present in the standard mixture. It shows a low intensity molecular ion peak at rnle 300 and a strong M - CH2I peak at mle 159. The ion fragment peak a t 243 has been found to be characteristic of 2-alkyl alcohols and may represent the fragment
L
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ANALYTICAL CHEMISTRY, VOL. 47, NO. 12, OCTOBER 1975
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ANALYTICAL CHEMISTRY, VOL. 47, NO. 12, OCTOBER 1975
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The reaction of the alcohols to form IMDMS ethers is typical of the reaction described for similar derivatives of steroids ( 3 , 4 ) . From the mass spectrum of the derivatized acids (Figures 2 and 3, spectra A and B ) , it is evident that iodomethyltetramethylmethyldisiloxane (IMTMMDS) esters were formed with the acids. A weak molecular ion was present in a-hydroxyisovaleric acid but was missing in lactic acid. Additionally, the hydroxyl group of lactic acid was not derivatized as shown by the mass spectrum of lactic acid (Figure 2 , spectrum A ) , perhaps because the acidic characteristic of the acid proton in lactic acid is increased by the presence of the a-hydroxyl group and BMDCS may preferentially react with the more acidic proton first. Once the bromomethyltetramethylmethyldisiloxane (BMTMMDS) ester derivative is formed, two factors greatly reduce the reactivity of the hydroxyl proton: 1)reduced acidity due to the formation of the ester and 2) steric hindrance effect due to the large BMTMMDS group. An intense molecular ion peak was present in the spectrum of the IMTMMDS ester of aryl benzoic acid a t mle 408 (Figure 3, specturm B ) , but little or no molecular ion was detected with alkyl acid esters. The spectra of the acids contained relatively intense M - CH2I and M - CH3 fragments. In some instances M - I was detected. There were several fragments derived from the IMTMMDS portion of the ester that was present in all the acid spectra (Figures 2 and 3, spectra A and B ) . These fragments were a t rnle 273, 245, 163, 131, and 117. The fragment at rnle 163, although present, is too weak to show in the graph of the spectrum of the IMTMMDS ester of benzoic acid (Figure 3, spectrum B ) . The possible structures of the fragments are as follows:
1
:H3
