1266
Anal. Chem. 1986, 58, 1266-1269
Determination of Thiols and Selenols by Titration with Methylmercury with End Point Detection by Nuclear Magnetic Resonance Spectrometry Dallas L. Rabenstein,*' R. Stephen Reid,2 Garry Yamashita, Khoon-Sin Tan, and Alan P. Arnold3 Department of Chemistry, University of Alberta, Edmonton, Alberta, Canada T6G 2G2 With the widespread application of nuclear magnetic resonance (NMR) spectrometry to the study of biological molecules, it is of interest to develop methods with which they can be characterized directly in the NMR tube, e.g., methods with which functional groups can be determined. In this paper, we describe methods for the determination of thiol and selenol groups in such molecules by titration with methylmercuric hydroxide (CH,HgOH) with end point detection by NMR. CH3HgOH is added directly to the sample in the NMR tube. The end point is detected by changes in the chemical shift of a resonance from an indicator molecule, which reacts with CH3HgOH added after the titration of thiol or selenol groups is complete. The methods are illustrated by results from the titration of the thiol groups of glutathione and hemoglobin and the selenol group of selenocysteamine. In the titration of hemoglobin, the interfering hemoglobin resonances are eliminated by using the spin echo Fourier transform NMR method (1).
EXPERIMENTAL SECTION Chemicals. Methylmercury(I1) iodide was from Alfa Division, Ventron Corp. Cysteamine, ergothioneine, glutathione, and thiouracil from Sigma and tetramethylammonium hydroxide from Eastman Organic Chemicals were used as received. Selenocysteamine was prepared from selenocystamine (Sigma) by electrolytic reduction over a mercury pool electrode (2). Preparation of Titrant Solution. Stock solutions of CH3HgOH were prepared from CH3HgI as previously described (3). The concentrations of the stock solutions were determined by titration with chloride using potentiometric end point detection (4) or by titration with thiosulfate using Michler's thioketone as a visual indicator (5). The concentrations were in the range 0.15-0.17 M. For most titrations, titrant solutions were prepared by tenfold dilution of the stock solutions. Because of the toxicity of CHSHg", the concentrated stock solutions were opened only in the fume hood and handled with gloves. Preparation and Assay of Hemoglobin Solution. Venous blood collected in Vacutainers (Becton, Dickinson and Co.) containing EDTA solution was centrifuged at 5000 rpm at 4 O C for 15 min, the plasma and buffy coat removed, and then 10 mL of packed cells was hemolyzed by sonication (Heat SystemsUltrasonics Model W225R sonicator) for 30 s. The hemolyzed erythrocytes were then centrifuged at 20000 rpm for 1h to remove cell debris. The resulting solution was placed in a dialysis bag (Spectrapor dialysis tubing, MW cutoff 6000-8000) with 0.2 g of CPG-lipoamide beads (Pierce Chemical Co.) and dialyzed against 2 x 200 mL of D20 phosphate buffer, pD 7.0, for 12-h periods. The resulting solution gave no indication of a significant (>0.2 mM) concentration of any small molecule, as judged by the absence of resonances in the spin echo Fourier transform spectrum obtained with a between-pulse delay time of 0.060 s (I). The hemoglobin concentration and the percentage of methemoglobin were determined to be 2.06 mM and 2%, respectively, by a standard spectrophotometric assay (6); for comparison, the percentage of methemoglobin in vivo is 1-3% (6). The solution prepared by the above procedure is not a pure hemoglobin solution; however under the titration conditions Present address: Department of Chemistry, University of California-Riverside, Riverside, CA. Present address: Department of Chemistry, University of Saskatchewan, Saskatoon, Saskatchewan, Canada. Present address: Department of Chemistry, University of Tasmania, Hobart, Tasmania, Australia. 0003-2700/86/0358-1266$01.50/0
CH3HgOH binds first to thiol groups then to the indicator and finally to other functional groups so that the only potential interferences are other thiol-containing molecules. The only other thiols present at significant concentrations relative to that of hemoglobin are glutathione and ergothioneine (7), both of which are removed by dialysis. NMR Measurements. IH NMR spectra were measured at 400 MHz with a Bruker WH-400/DS spectrometer or at 360 MHz with a Bruker WM-360 spectrometer. In the titration of selenocysteamine and glutathione, spectra were measured by the standard single pulse method. In the titration of hemoglobin, spectra were measured by the spin echo Fourier transform (SEFT) using a 7 of 0.060 s to eliminate method (90°-~-1800-~-acquisition) protein resonances ( I ) . Typically 50 transients were collected for each spectrum, using a recycle time of 2-3 s. Chemical shifts were measured relative to the methyl resonance of tetramethylammonium ion (TMA) (3.176 ppm relative to the methyl resonance of sodium 4,4-dimethyl-4-silapentanesulfonicacid (DSS)) or the methyl resonance of tert-butyl alcohol (1.2365 ppm). The titrations were generally done on D20solutions to avoid the dynamic range problem encountered when measuring spectra of dilute H20 solutions by pulse FT NMR methods. However, some titrations were done on H20 solutions, and the H20 resonance was suppressed with a presaturation pulse at the frequency of the H20resonance (8). Since the indicator resonances are far removed from the H20 resonance, titrations can easily be done on H20 solutions using this and other methods to avoid the dynamic range problem (9). With protein solutions,the dynamic range problem can also be avoided by taking advantage of the attenuation of the H20 resonance by spin-spin relaxation (IO). Titration Procedure. The thiol or selenol is titrated directly in the NMR tube. The procedure involves adding a carefully measured volume of sample solution, generally in the 0.5-mL range, and 25 p L of solution containing 0.05 M indicator (ergothioneine or thiouracil)and 0.05 M chemical shift reference (TMA or tert-butyl alcohol) to the NMR tube. Titrant (10 or 20 pL) is then added; the contents of the NMR tube are carefully mixed, and the spectrum is measured. This procedure is repeated until enough points have been obtained beyond the end point to define the titration curve. Titrant can be conveniently added by using a calibrated 10-pL syringe.
RESULTS AND DISCUSSION Analytical Reactions. The analytical reactions are
RSH
+ CH,HgOH
RSeH
+ CH,HgOH
+ H20 RSeHgCH3 + H 2 0
e RSHgCH,
(1)
(2)
Thiol and selenol groups have a high affinity for CH3Hgn, with formation constants in the ranges of 1016-1017 (3, 5) and 1016-1018 (11),respectively. CH3Hg" also forms complexes with other functional groups in peptides and proteins and with ions commonly present in biological fluids (12). The logarithms of the conditional formation constants, K,, for model complexes of thiols, selenols, and other potential binding sites are shown as a function of pH in Figure 1. These data indicate that titration of thiol and selenol groups will be complete before CH,Hgrl reacts with other functional groups or with chloride or iodide. Indicators. Ergothioneine (I) and thiouracil (11) have both been used as NMR indicators to locate the end points of the titration. Curve C in Figure 1 indicates that ergothioneine satisfies the requirement that the conditional formation constant of the CH3HgrLindicator complex be less than those 0 1988 American Chemical Soclety
ANALYTICAL CHEMISTRY, VOL. 58, NO. 6, MAY 1986
(CH
+ J,N CH COT I
CHI "C=C I
I
15.0
I
*C N'H
N
/
1 7
t-
1
\
13.0
1267
I
SH 11.0
-
v 9.0
-
z
0
0
0
A
7.0
of the CH,Hg"-thiol and selenol complexes but larger than those of CH3HgI1complexes of the other potential binding sites. Although Kf, for the CH3Hg"-thiouracil complex has not been determined, it is probably similar to that for the CH3Hg11-ergothioneine complex, since the sulfur in both molecules is in a thiol/thione equilibrium. Also, the results of titrations using thiouracil as indicator indicate the conditional formation constant of its CH,Hg" complex to be of an appropriate magnitude. The resonance for the carbon-bonded ring proton of ergothioneine is a singlet at 6.78 ppm, while the two carbon-bonded protons of thiouracil give two doublets, centered at 5.99 and 7.62 ppm. The chemical shifts change upon reaction with CH,Hg1I. Exchange of indicator between free and CH,Hg" complexed forms is fast on the NMR time scale so that exchange averaged resonances with chemical shifts given by eq 3 are observed. In eq 3 6o is the chemical shift of the exchange 60
= fC@C - 6f)
+ 6f
5.0
-
3.0
-
1.0
-
\-4.0
6.0
PH
8.0
10.0
Figure 1. pH dependence of the conditional formation constants (KfJ for the CH,Hg" complexes of (A) selenocysteine (selenium), (B) glutathione (sulfur), (C) ergothioneine (sulfur), (D) iodide, (E) chloride, (F) N-methyllmidazole, (G) glyclne (nitrogen), (H) acetylglyclne (oxygen), and (I) methioneine (sulfur). K,, is for the reaction CH,Hg, 4- L, F? CH,HgL, where CH,Hg, and L, represent all the free forms of methylmercury and ligand and CH,HgL, all the complexed species (3). 6.86,
I
I
!
I
I
I
I
,
i
(3)
averaged resonance, 6, and 6fthe chemical shifts of the complexed and free indicator, and f, the fraction of the indicator that is complexed with CH3Hg". According to eq 3, the chemical shifts of indicator resonances will remain constant up to the end point and then will shift linearly as CH,Hg" is added beyond the end point. It is important to note that bo depends on the fraction and not the concentration of the complexed indicator, and thus volume corrections are not necessary. Titration of Thiols. The method was evaluated by titration of a pD 7.41 phosphate buffered D 2 0 solution of glutathione. Aliquots (0.5 mL) of solution were placed in NMR tubes; 20 pL of phosphate buffered solution containing 50 mM ergothioneine and 50 mM TMA were added, and the samples were then titrated with 0.016 46 M CH3HgOD. 'H NMR spectra were measured after each addition of titrant, and the chemical shift of the resonance for the carbon-bonded ring proton of ergothioneine (resonance e2) was monitored. The NMR tubes were weighed before and after addition of the glutathione solution, and the actual volume of each aliquot titrated was determined by using a value of 1.1044 g/mL for the density of the D 2 0 solution. A typical titration curve is shown in Figure 2. As predicted by eq 3 and the conditional formation constants in Figure 1,. the chemical shift of the ergothioneine resonance remains constant at the chemical shift of free ergothioneine until the glutathione has been titrated, and then it shifts linearly as
6.78
'
0
I
1
,
40
80
120
1 160
Volume of CH3Hg ( 11 ), pP
Flgure 2. Chemical shift of resonance e2 of ergothlone during the titration of -3 mM glutathione In pD 7.4 phosphate buffered D20 solution wlth 0.01646 M CH,!-IgOD. From the end point in this titration, the concentration was determined to be 2.88 mM.
more CH,HgOH is added. An average concentration of 2,76 mM with a standard deviation of 0.09 mM was obtained from six titrations. For comparison, the concentration was also determined by reaction of the thiol group with iodoacetamide, followed by titration of the displaced protons with base, to be 2.795 mM (13). Application to the titration of thiol groups in proteins is demonstrated by titration of the hemoglobin solution. Ali-
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ANALYTICAL CHEMISTRY, VOL. 58, NO. 6, MAY 1986
li
1
7.0
6 0
5 0 PPrn
4.0
3.0
Flgure 3. ’H NMR spectrum (400 MHz) for a solution containing 1.96 mM hemoglobin, 2.4 mM ergothionelne, and 2.4 mM TMA in pD 7.4
D,O-phosphate buffer. The spectrum was measured by the spin echo pulse sequence with a between-pulse delay of 0.06 s. quots (0.500 mL) of the phosphate buffered D 2 0 solution of hemoglobin were placed in NMR tubes; sufficient ergothioneine and TMA solution was added t~ give a concentration of -2 mM, and the samples were titrated in the NMR tubes with CH3HgOD. Figure 3 shows an example of the lH NMR spectra that were obtained during the titration by the spin echo Fourier transform method ( 1 ) . The intense protein resonances are completely eliminated; however, resonances e l and e2 of ergothioneine are only slightly reduced in intensity due to spin-spin relaxation. Resonace e3 is considerably reduced in intensity by phase modulation that occurs during the spin echo pulse sequence ( I ) . Titration curves for the hemoglobin titration are identical in appearance with that shown in Figure 2. An average thiol concentration of 4.01 mM with a standard deviation of 0.05 mM was determined for the hemoglobin solution. Combining this with the hemoglobin concentration determined spectrophotometrically gives 1.95 mol of SH/mol of hemoglobin, as compared to the accepted value of 2 (7). Conflicting reports have appeared regarding the time required for reaction of CH3Hg” with protein thiol groups (14-16)- In the method described here, slow binding of titrant to thiol groups can be detected easily by continuous monitoring of the chemical shift of the indicator resonance once the end point has been passed. For example, in the present case, the matter was quickly settled by measuring spectra as a function of time after the last aliquot of titrant had been added. Any slow uptake or release of CH3Hgn by the protein would result in a shift in the indicator resonance; no shift was observed over a period of several hours. Titration of Selenols. The conditional formation constants in Figure 1 for the CH3Hg11-selenocysteine complex indicate that CH3Hgnwill react completely with selenol groups before reacting with other potential binding sites. This was confirmed by titration of a selenocysteamine solution containing 3 mM thiouracil with 0.1478 M CH3HgOH. The titration curve obtained by monitoring the thiouracil resonance a t -7.63 ppm was similar to that shown in Figure 2, and a selenocysteamine concentration of 18.18 mM was obtained from the end point. Comparison to Other Methods. A variety of methods have been developed for determining thiol groups in biological molecules (17); however, very few methods have been described for the determination of selenols (18). Spectrophotometric methods that are based on measuring the absorbance of 2-nitro-5-thiobenzoic acid formed by reaction of thiol with 5,5’-dithiobis(2-nitrobenzoicacid) (DTNB) are among the
most widely used methods for the determination of thiol groups (19,ZO). However, because the absorbance is measured a t 410 nM, it is difficult to determine the thiol groups of heme-containing proteins by the DTNB method. In comparison, the thiol groups in hemoglobin can readily be determined by the NMR titration method described here. In fact, the thiol content of hemolyzed red blood cells can be determined directly by this method by using the spin echo pulse sequence to eliminate the interfering protein resonances (21) and the ergothioneine naturally present in red blood cells to indicate the titration end point. Because of the selectivity of the reaction of CH3HgOH with thiols (Figure 1)and the lack of interference from other sample constituents in the NMR measurement, the NMR titration method would appear to be superior to other methods for determining the thiol content of heme-containing proteins, red blood cells, and other highly colored samples.
CONCLUSIONS The titrations described in this paper are easy to perform and can be used to determine thiol and selenol groups in biological molecules ranging in size from amino acids to proteins. With the conditions used to obtain the spectrum in Figure 3, it took -2 min of spectrometer accumulation/ point on the titration curve. After the first titration, successive titrations can be done more quickly by measuring spectra at only two or three points before the end point. Although a sample volume of -0.5 mL was used, NMR spectra can be conveniently obtained from volumes as small as -0.2 mL. With the indicator and titrant concentrations used here, -0.2 pmol of thiol or selenol could be titrated with an end point volume of 10 pL. Even smaller quantities can be titrated by using smaller titrant and indicator concentrations. The lower limit will depend on the sensitivity of the spectrometer. The titration described here can also be used to easily block, in the NMR tube, the thiol or selenol groups of biological molecules whose chemistry is being studied by IH NMR. Since any excess CH3HgOH added reacts with indicator, the thiol or selenol groups are selectively blocked. Registry No. MeHe, 16056-34-1;ClHgMe, 115-09-3;IHgMe, 143-36-2; glutathione, 70-18-8; selenocysteamine, 21681-94-7; selenocysteine-MeHgn complex, 88722-09-2;glutathione-MeHg” complex, 42166-99-4;ergothioneine-MeHgncomplex, 100333-90-2; N-methylimidazole-MeHgn complex, 65149-20-4;glycine-MeHe complex, 61113-44-8; acetylglycine-MeHg” complex, 40187-68-6; methionine-MeHg” complex, 54517-54-3;ergothioneine,497-30-3. LITERATURE CITED (1) (2) (3) (4)
(5) (6) (7) (8)
(9)
(IO) (11) (12) (13) (14) (15) (16) (17) (18)
(19) (20)
Rabensteln, D. L. Anal. Chem. 1978, 5 0 , 1265A-1276A. Saetre, R.; Rabenstein, D. L. Anal. Chem. 1978, 50, 276-280. Reid, R. S.; Rabensteln, D. L. Can. J . Chem. 1981, 5 9 , 1505-1514. Rabensteln, D. L.; Fairhurst, M. T. J . Am. Chem. SOC. 1975. 97, 2086-2092. Arnold, A. P.; Canty, A. J. Can. J . Chem. 1983, 67, 1428-1434. Makarem, A. I n “Clinical Chemistry”; Henry, R. J., Cannon, D. C., Winkelman, J. W., Eds.; Harper and Row: New York, 1974, pp 1131-1135. Jocelyn, P. C. “Biochemistry of the SH Group”; Academic Press: New York, 1972. Jesson. J. P.; Meakin, P.; Knlessel, 0. J . Am. Chem. SOC.1973. 9 5 , 618-620. Hore, P. J. J . Magn. Reson. 1983, 55, 283-300. Rabenstein, D. L.; Isab, A. A. J . Magn. Reson. 1979, 36, 281-286. Arnold, A. P.; Tan, K.-S.;Rabensteln, D. L. Inorg. Chem., in press. Rabenstein. D. L. Acc. Chem. Res. 1978, 7 7 , 100-107. Benesch, R.; Benesch, R. E. Biochim. Biophys. Acta 1957, 23. 643-644. Leach, S. J. Aust. J . Chem. 1980, 73,520-546. Leach, S.J. Aust. J . Chem. 1980, 73,547-566. Forbes, W. F.; Hamlin, C. R. Can. J . Chem. 1988, 46, 3033-3040. Ashworth, M. R. F. “The Determination of Sulfur-Containing Groups”; Academic Press: New York, 1976; Vol. 2. Shamberger, R. J. “Biochemistry of Selenium”; Plenum: New York, 1983. Ellman, G. L. Arch. Biochem. Biophys. 1959, 82, 70-77. Beutler, E.; Duron, 0.;Kelly, B. M. J . Lab. Clin. Med. 1963, 67, 882-888.
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Anal. Chem. 1986, 58, 1269-1272 (21)
Rabensteln, D. L. J . Blochem. Blophys. Methods 1984, 9 , 277-306.
RECEIVED for review June 6, 1985. Accepted November 25, 1985. This research was supported by an operating grant to D.L.R. from the Natural Sciences and Engineering Research
I
Council of Canada and by the University of Alberta. An I. W. Killam Scholarship to R.S.R., an Alberta Heritage Foundation for Medical Research scholarship to K.S.T., and an AHFMR postdoctoral fellowship to A.P.A. are gratefully acknowledged.
Ion Mobility Spectrometry/Mass Spectrometry of Some Prescription and Illicit Drugs A. H. Lawrence Unsteady Aerodynamics Laboratory, National Aeronautical Establishment, National Research Council of Canada, Ottawa, Ontario, Canada K1A OR6 Ion mobility spectrometry (IMS), also known as plasma chromatography, is an analytical technique that distinguishes ionic species on the basis of the differences in the drift velocity through a gas under an applied electrostatic field (1). It is a sensitive technique for detection of trace organics under atmospheric pressure conditions. Experimental results are usually reported in terms of ion mobility reduced to standard temperature and pressure KO (cm2 V-l s-'). The IMS has been commercially available for about 10 years but has received moderate attention as an analytical laboratory tool. Recently, however, there has been renewed interest in IMS as evidenced by the number of publications in the scientific literature (2-9). Moreover, the instrument meets a wide range of performance and operational requirements-good sensitivity, fast response time, operation at atmospheric pressure-and is rapidly gaining acceptance as a field instrument ( 1 0 , I I ) . Work is in progress in this laboratory to develop IMS-based technology, specifically designed for law enforcement and forensic field applications. We have recently reported on the application of surface sampling and IMS analysis to the detection of organonitrate explosives and drug residues. These studies addressed the effect of matrix and potential interfering chemicals on the discrimination and detection capability of the IMS (12,13). As well, in these investigations, reduced mobilities (KO) were used as the qualitative measurement of specific ions; however, a more direct identification method and the obvious way to assign masses to ions giving particular mobility peaks is to interface the IMS with a quadrupole mass spectrometer (MS). The present paper describes the identification of the primary ions associated with the mobility peaks of several prescription and illicit drugs using ion mobility spectrometry/mass spectrometry (IMS/MS).
EXPERIMENTAL SECTION Reagents. Linde high-purity air was dried by Linde molecular sieve 13X and used for both carrier and drift gases. All drugs were obtained from the reference collection of Health and Welfare Canada and were used without further purification. Samples of amphetamine, methamphetamine, methylenedioxyamphetamine, and N-acetylamphetamine were prepared as lo4 g/mL solutions in ether. The remainder of the drugs were prepared as g/mL solutions in methanol. Apparatus. The data presented in this paper were collected with a Phemto-Chem MMS-160 IMS/MS (PCP, Inc., West Palm Beach, FL). The instrument has been described elsewhere (14). The experimental parameters used to operate the IMS/MS are tabulated in Table I. Total ion mobility spectra were obtained by gating grid G2with grid GI held continuously open; the IMS electrometer detector is used to obtain mobility data, and the quadrupole mass filter is not used. Mass-identified ion mobility spectra were obtained by 0003-2700/86/0358-1269$01.50/0
Table I. Experimental Conditions parameter
value
Ion Mobility Spectrometer drift length (between grid G2and IMS collector) drift voltage carrier gas (purified air) drift gas (purified air) inlet temperature drift temperature pressure dwell time gate width delay timea digitizer resolution
5cm
.
+2800 V 100 cm3/min 500 cm3/min 210 OC
220 OC atmosphere 20 gslchannel 0.2 ms 1.28 ms 9 bits
Mass Spectrometer pressure resolution scanning speed sensitivity
9.5 x
torr 850 (range 0-1000) 1000 amu/s 200 x lo2
"Time between gate opening and start of data collection. operating the instrument with the grid gating procedure described above and with the mass spectrometer tuned to a specific m l t value. Since the detector sees only one ionic species, the mobility spectrum consists of only that peak corresponding to the ion. Furthermore, the drift time is slightly longer than that observed with the previous mode, since the ion lens and the orifice interfacing of the mass spectrometer added extra length to the drift space. Finally, mass spectral data were obtained by holding both grids G1 and G2open, allowing all the ions formed in the IMS to drift down through the tube and into the mass spectrometer. The data obtained with the Phemto-Chem MMS-160 instrument were taken by signal averaging a given number of 20-ms scans in a Nicolet signal averager (FT 1072, Nicolet Instrument, Inc.). The resulting ion mobility spectrum was displayed on an x-y recorder. All samples were introduced into the inlet of the IMS using a clean stainless steel wire; the wire was dipped in the drug solution and the solvent air evaporated prior to sample introduction.
RESULTS AND DISCUSSION The positive ion mobility spectra of all the drugs investigated, with the exception of phencyclidine, were simple with no appreciable fragments or ion clusters. The majority of compounds examined produced a single main ion peak corresponding to Mf or [MH]+ ion (Table 11) where M is the molecular species. It is well-established that, when nitrogen or air containing a little water is used as a carrier gas, positive 0 1966 American Chemical Society
