2533
Anal. Chem. 1987, 59, 2533-2534
CORRESPONDENCE Mixed-Mode Column Thermospray Liquid Chromatography/Mass Spectrometry Sir: Thermospray liquid chromatography/mass spectrometry (LC/MS) typically involves the use of reversed-phase columns and volatile buffer salts such as ammonium acetate (1-3). However, these simple reversed-phase conditions may not provide sufficient separations of mixtures of ionic or neutral and ionic compounds (4). Ion-pair reagents such as alkylsulfonatesmay provide improved chromatographic results (4) but these nonvolatile salts are not compatible with the thermospray ion source (1). We have developed a strategy for thermospray LC/MS analysis of mixtures that contain ionic compounds that are not separated by simple reversed-phase chromatography. This approach utilizes a commercially available mixed-mode hydrophobic ion-exchangeHPLC column that simulates ion-pair separations (5, 6). The column incorporates C-4 alkyl and phenylsulfonate functionalities bound to a silica support. A mobile phase of ammonium trifluoroacetate (1M, pH 2.5) and methanol (7525) provides good chromatographic results with this column and is volatile under the thermospray conditions. In order to demonstrate the general utility of this strategy, we have chosen to examine a mixture of the active ingredients in common cold remedies (Table I). These compounds are present in the acidic mobile phase as a mixture of neutral and ionic species. Separation of some of these compounds by ion-pair high-performance liquid chromatography (HPLC) has been previously reported (7).
Table I. Thermospray LC/MS Data for Compounds 1-5 (Ionization Mode: Discharge On) relative intensity compound
molwt (M + H)' 179
4,
8
(M+ NH4)'
(M+HH20)'
100
(mlz 180)
%HI
1
2 HO-CH-CH2-NH-CHs
b,
167
100
(mlz 168)
11
(mlz 150)
3 :HZ HO-CH-CH-CHI
151
b
100
(mlz 152)
12
(mlz 134)
4
EXPERIMENTAL SECTION Reagents. Phenacetin (1),guaifenesin (3-(2-methoxyphenoxy)-1,2-propanediol,2), phenylephrine (3), norephedrine (4), and ephedrine (5) were obtained from Aldrich Chemical Co. Ammonium hydroxide (MaJIinckrodt,Inc.), trifluoroacetic acid (Fisher Scientific),and methanol (Burdick and Jackson) were purchased and used without further treatment. Water was obtained from a Millipore Milli-Q purification system. Instrumentation. The HPLC system consisted of a PerkinElmer Series 4 solvent delivery system, a Rheodyne 7126 injector with a 50-pL loop, an RP-SCX column (5-pm particle size, 100-8, pore size, 4.6 mm X 25 cm; ES Industries, Marlton, NJ), and an LDC Spectromonitor I11 UV detector which was equipped with a max-N high-pressure cell (LDC). The exit line from the detector was connected directly to a thermospray interface (Vestec Corp., Houston, TX) which was fitted to a Finnigan 3000 mass spectrometer. On-line data reduction was performed by a Finnigan INCOS data system. The discharge mode of ionization was employed ( 2 , 4 ) . The temperatures for the thermospray interface were TI= 115 O C , T2= 195 O C , and block = 350 O C . Procedures. The mobile phase was a 75:25 mixture of 1 M ammonium trifluoroacetate and methanol. The ammonium trifluoroacetate solution was prepared by diluting 160 mL of trifluoroacetic acid and 188 mL of concentrated ammonium hydroxide to 2 L with water (pH 2.5). The chromatographic flow rate was 0.5 mL/min. Sample injections consisted of ca. 1pg of each comDonent. RESULTS AND DISCUSSION
Good separation of the components of a mixture of 1-5 was achieved by use of the mixed-mode column (Figure 1). In the acidic chromatographic mobile phases, phenacetin (I) and guaifenesin (2) are neutral solutes, while phenylephrine (3), 0003-2700/87/0359-2533$01.50/0
165
y 3
HO-CH-CH-NH-CH,
100
(mlz 166)
4
(mlz 148)
5
norephedrine (41, and ephedrine (5) are protonated species. The order of elution of phenacetin (1) and norephedrine (4) from the mixed-mode column were reversed in comparison to ion-pair HPLC with pentanesulfonate or hexanesulfonate reagents (7). The detection of compounds 1-5 was achieved by thermospray MS with discharge-on ionization (2,4). Quasi-molecular (M + H)+ ions were observed for all of the solutes (Table I). No fragmentation was observed for compounds 1 or 2. An (M H - H20)+ion of low intensity was observed for compounds 4 and 5 which contain benzylic alcohol groups. The choice of mobile phase composition and concentration was somewhat arbitrary. Other volatile salts such as ammonium acetate should also be satisfactory, but they were not investigated. The 0.05-0.1 M salt concentrations which are normally used for reversed-phase thermospray LC/MS were too low for acceptable chromatography of the model compounds with the mixed-mode column. No attempt was made to determine the minimum ionic strength which was required for acceptable chromatographic results. An ionic strength of 1 M was chosen because this concentration was assumed to represent an approximate upper limit for the requirements of the mixed-mode column. The present mobile phase did not cause any problems with the thermospray vaporizer.
+
@ 1987 American Chemical Society
Anal. Chem. 1987, 59, 2534-2535
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of mixtures of ionic or neutral and ionic compounds which are not well separated by simple reversed-phase HPLC. Registry No. 1,62-44-2; 2,93-14-1;3, 59-42-7;4, 700-65-2;5, 299-42-3; ammonium trifluoroacetate, 3336-58-1.
4
3 1
I
LITERATURE CITED
10
15 MINUTES
20
(1) Garteiz, D.; Vestal, M. LC Mag. 1982, 334. (2) Voyksner, R.; Haney, C. Anal. Chem. 1986, 57, 991. (3) Covey, T.; Lee, E.; Bruins, A.; Henlon, J. Anal. Chem. 1966, 58, 1451A. (4) Snyder, L.; Kirkland, J. Introductlon to Madern UquM Chromatography; Wiiey: New York, 1979; Chapter 11. (5) Crowther. J.; Hartwick, R. Chromatographie 1982, 16, 349. (6) Floyd, T.; Clcero, S.;Fazb, S.;Ragilone, T.; Hus, S.; Winkle, S.;Hartwick, R. Anal. Blochem. 1986, 154, 570. (7) Bidlingrneyer, B. J. Chromatogr. Scl. 1980, 18, 525.
' Current address:
Finnigan-MAT, Rockville, MD.
Flgwe 1. Reconstructed ion chromatogram of a mixture of compounds 1-5.
However, the vacuum line between the thermospray source and the rough pump was found to clog with salt after a few hours of operation. Therefore, the HPLC column flow rate was limited to 0.5 mL/min. CONCLUSION Mixed-mode column thermospray LC/MS (discharge ionization) appears to offer an alternative approach to the analysis
John R. Lloyd' Mary Lou Cotter David Ohori Alan R. Oyler* Research Laboratories Ortho Pharmaceutical Corporation Raritan, New Jersey 08869-0602
RECEIVED for review March 11,1987. Accepted June 29,1987.
AIDS FOR ANALYTICAL CHEMISTS Automated Sample Cell Cleaner D. E. Bautz and J. D. Ingle, Jr.* Department of Chemistry, Oregon State University, Corvallis, Oregon 97331 The majority of spectrophotometricand spectrofluorometric measurements are made on solutions in 1 cm path length sample cells. Many analytical procedures are based on monitoring the formation of an absorbing or fluorescing product by reaction of the analyte with suitable reagents. To simplify the procedure and to increase sample throughout, the sample and reagent solutions can be added to and mixed in a sample cell that is secured in a sample-cell holder. Addition of samples and reagents with automatic pipets (e.g., Eppendorf) or automatic syringe injectors has proved to be simple and rapid (I). Normally, one evacuates the contents of the cell with a vacuum aspirator and rinses several times with a blank solution from a wash bottle to prepare the cell for the next sample or standard. In continuous flow analysis systems, the sample flow cell is cleaned between sample plugs by the carrier stream without operator attention. With rapid and often automated addition of samples and reagents to the sample cell and microcomputer-based data acquisition, calculations, and reporting, we find that the emptying and cleaning of the sample cell between measurementa is the most time-consuming and tedious step during the analytical measurement. To address these limitations, a microcomputer-controlled cell cleaner was designed to perform the function of cell cleaning. This device allows spectrometric measurements with a conventional cell to be performed in a more automated fashion and frees the operator for more important tasks. The actual size of the cell cleaning system is small enough to be placed adjacent to or directly on top of the instrument
Table I. Instrumental Components
component
model
source
Dion ex,
three-way solution valve and pneumatic activator
30520
solid-state relays
CA 7052-04-B04-F Grayhill,
three-way air valve
MBD-002
Pump
Sunnyvale, LaGrange, IL Skinner, New Britain, CT Lab pump junior Fluid Metering, Inc., Oyster Bay, NY
containing the sample cell. The cell cleaner was used here to clean a fluorometric cell containing quinine sulfate (QS) and the resulting blank signal was measured as an indicator of the completeness of cleaning. INSTRUMENTATION A schematic of the cell cleaning device is shown in Figure 1 and the components are specified in Table I. A glass capillary tube (0.125 in. i.d.) is glued along an inside edge of the sample cell with the bottom end of the tube located -1 mm above the bottom of the cell. The other end of the capillary tube is connected with 0.125 in. i.d. Telfon tubing to the common input port of a three-way solution valve. The other two ports are connected to a pump and to vacuum. The pump is turned on and off by switching the ac power to the pump with a solid-state relay (SSR1) controlled by TTL logic
0003-2700/87/0359-2534$01.50/00 1987 American Chemical Society
