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Anal. Chem. 1982, 5 4 , 451-456
Determination of Sulfa Drugs in Biological Fluids by Liquid Chromatography/Mass Spectrometry/Mass Spectrometry Jack D. Henlon” Dlagnostlc Laboratory, NYS College of Veterinary Medicine, Cornell Unlversiv, Ithaca, New York 14853
Bruce A. Thomson Sclex Inc., Thornhlll, Ontario, Canada
Peter H. Dawson Physics Dlvlslon, National Research Councll, Ottawa, Ontario, Canada
The technique of direct liquid introduction (DLI) liquid chromatography/mass spectrometry/mass spectrometry (LC/ MS/MS) is implemented on an atmospheric pressure chemlcal ionlzatlon (API ) triple quadrupole mass spectrometer. Experimental details are described. A series of sulfa drugs lncludlng sulfamethazine (ISM), sulflsoxazoie (SOX), suifadlazine (SD), and suifadimethoxlne (SDM) are studied in the full scan LC/MS, full scan LC/MS/MS, and selected ion monltorlng (SIM) LC/MS/MS modes. Comparisons between the data are made. Full scan LC/MS/MS and SIM LC/MS/MS results are described for the analysis of a raw organic extract of racehorse urine and plasma containing residues of administered suifadlmethoxlne. The method minimizes sample cleanup, protects the mass spectrometer from abuslve exposure to high levels of endogenous materials that result from direct Insertion probe sample Introduction, and provides retention t h e lnformatlon for the eluted components of Interest. On-column full scan sample detection llmlts in the low mlcrogram range were easily obtained using the DLI split effluent LC/MS/MS interface and could be increased by utilizing micro LC techniques and improved effluent desoivatlon techniques.
The analysis of polar, labile, and involatile drugs in complex chemical matrices still remains a challenging analytical problem in certain instances. Several groups have reported that the relatively new technique of MS/MS, or tandem mass spectrometry (1-3) can provide the capability of identifying unknown substances in such matrices without any sample pretreatment whatsoever (4). This is a very entjcing claim, but the implications often oversimplify the experimental facts involved with such experiments. For example, the introduction of high levels of endogenous components present in “raw” blood or urine into the conventional ion source of a tandem mass spectrometer constitutes an abuse to the optimum performance of the system. While a few such samples may be analyzed in this manner, it appears that screening hundreds of such samples between ion source cleanings is not practical. This problem is somewhat reduced through the use of an atmospheric pressure chemical ionization (API) source (5) of the type used in the present experiments which has proven to be less susceptible than a conventional source to memory effects and contamination problems. Nevertheless, it is clear that in many situations efficiency in the analysis is best served by performing a fast sample cleanup in order to eliminate the majority OF the matrix material before direct probe analysis. Such a cleanup also minimizes matrix effects encountered in quantitation, and usually results in higher 0003-2700/82/0354-0451$01.25/U
sensitivity toward the components of interest. Although the identification of trace or foreign substances by MS/MS directly from “raw” samples or crude extracts desorbed from a direct probe would be highly desirable and has received the most emphasis in previous publications, the inclusion of some chromatographic separation greatly increases the probability of successful identification. For example, rapid screening by direct probe of numerous untreated blood and urine samples may be feasible if a particular drug or compound is suspected. However, if one or more unknown foreign substances are present at low levels in the mixture, the analyst has no a priori way of knowing which ions are of interest and effective screening by MS/MS becomes a much more challenging problem. In most laboratories today, effective extraction followed by thin-layer chromatography (TLC) (6), GC/MS (7), and LC/MS (8, 9) is the method of choice. GC/MS has become a well-established technique for trace component identification provided the samples are amenable to GC analysis by some means (10). LC/MS offers complementary capability to GC/MS because it can afford separation of polar, labile compounds under relatively mild conditions (8, 11). Both GC/MS/MS and LC/MS/MS are therefore expected to offer an attractive combination of chromatographic separation and mass spectral information, resulting in the ability in many cases to screen complex samples with a fast, relatively low resolution chromatographic separation followed by MS/MS. In particular, a limitation resulting from the direct liquid introduction (DLI) technique of LC/MS (12) is that the resulting mass spectra are often very simple and lack sufficient specificity for identification (9,13,14). Also, coeluting components will appear superimposed in the mass spectrum making interpretation difficult. The technique of LC/MS/MS offers the capability of providing collision-induced-dissociation (CID) spectra of the characteristic LC/MS ions for each of the components of interest. This produces a CID or LC/ MS/MS mass spectrum which can yield an added degree of specificity over conventional DLI LC/MS, and eliminate interferences from coeluting species, unless the components form the same parent ion in the source. The potential of API for LC/MS analysis was explored by Horning and others (5) using a somewhat different source than used here. Very good sensitivity was achieved. This report explores the potential of a more open API source design and describes our initial results with the combination of LC and MS/MS in the determination of sulfa drugs found in biological fluids.
EXPERIMENTAL SECTION The drug standards utilized in this work were obtained from commercially available sources. They were checked for purity 0 1982 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 54, NO. 3, MARCH 1982
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Figure 1. Schematlc outline of LCIMS probe and Interface to the atmospheric pressure ion source: (1) LC eluant inlet to probe; (2) outlet to waste; (3)LC/MS probe; (4) zero air carrier gas in, flow rate = 1 L/min; (5) glass Inlet tube, 22 mm diameter; (6) spray of microdroplets from probe orlflce; (7) vaporlzation section (8 mm diameter by 10 cm long) wrapped wlth Nlchrome heating wire; (8) stainless steel wire mesh for vaporization and desolvation; (9) corona discharge needle; (10) orifice Into vacuum chamber; (11) atmospheric pressure plenum chamber; (12) exhaust of carrier gas and vaporlzed material.
by TLC and LC before use, Acetonitrile was of distilled-in-glass quality and purchased from Burdick and Jackson (Muskegon,MI). Water was LC grade and purchased from J. T. Baker Co. (Phillipsburg, NJ). The plasma and urine extracts were obtained as extracts in 1:l:lhexane:dichloromethane:ether at pH 3.2 and 3.0, respectively, from a racehorse which had received an unknown dose of sulfadimethoxine. The extraction solvent was removed under a gentle scream of nitrogen at 65 "C and taken up in 250 pL of acetonitrile. The HPLC was a Waters, ALG-202 instrument consisting of two Model 6000A pumps, a M-660 solvent programmer, and a U6K loop injector equipped with a 65-pL sample loop (Waters Associates, Milford, MA). The LC column was a Whatman 4.6 mm X 25 cm PXS 10/25 ODS (Whatman, Clifton, NJ) connected to a Perkin-Elmer LC-55 variable-wavelength detector which was output to a Perkin-Elmer Model 023 10-mV recorder (PerkinElmer, Norwalk, CT). The UV detector was set at 254 nm for the sulfa drug analyses. The eluant flow rate was maintained at 1 mL/min and the gradient was linear from 10/90 to 90/10 CH3CN/H20 over a 10-min interval. The exit of the UV detector was connected to a HewlettPackard DLI LC/MS (13) probe interface (Hewlett-Packard, Co., Palo Alto, CA) via an in-line 0.4-pm filter (Rheodyne, Inc., Berkeley, CA). The LC/MS probe was inserted through a modified glass inlet of the API source of a Sciex TAGA 6000 triple quadrupole mass spectrometer (Sciex, Inc., Thornhill, Ontario, Canada). The ion source is designed to accept a sample inlet tube of up to 22 mm diameter through the front plate. The inlet tube constructed to couple the LC/MS probe is shown in Figure 1. The probe was supported in the 22 mm section with an O-ring clamp at one end and a wire spring (not shown) wrapped around the downstream end. The narrower section of tubing (8 mm diameter by 10 cm long) was wrapped with nichrome wire and thermally insulated with asbestos tape. A piece of stainless steel wire mesh (25 pm wire, 100 X 100 per in.) was inserted in the end of this section to facilitate desolvation and vaporization of the solute material in the microdroplets produced by the DLI LC/MS probe. A temperature of 300 "C was maintained at the wall of the vaporization section. In operation, a jet of microdroplets was established with the probe located outside the ion source, and then the probe was inserted into the inlet tube. A flow of carrier gas (Zero Grade Air, Matheson, East Rutherford, NJ) maintained at 1 L/min was introduced tangentially into the 22 mm section, providing a laminar flow of gas to transport the nebulized droplets into the vaporization tube. In bench testa the jet of droplets could be observed carried by the gas stream through the center of the tube without contacting the walls. The arrangement of heated glass and in-line stainless steel mesh appeared to offer no deleterous effects upon the samples or ion current chromatograms. However, the sensitivity limits experienced suggest that the desolvation process in this work was not optimum and needs more attention. Initial positive ion chemical
ionization (PCI) LC/MS experiments were conducted by admitting a solution of a representative sulfa drug, sulfamethazine (SM), into the API source via the DLI LC/MS probe interface. This was accomplished by pumping a 50 ppm solution of SM in 50% CH3CN/H20at a flow rate of 1mL/min through a column split (1O:l) to the LC/MS interface. After a suitable liquid "jet" was established through the 5-pm orifice in the removable diaphragm at the tip of the probe, the probe was inserted into the API source. In practice, it was found that the length of the jet could be varied from about 2 cm long, representing approximately 1% of the LC effluent, up to approximately 20 cm long, representing 10% of the total effluent flowing at 1mL/min. The length of the jet was not as critical a parameter as is the case in more conventional systems (13). With the solution of SM passing into the source, ion source and instrument parameters were tuned to provide maximum sample ion current signal. The position of the probe in the inlet tube and the horizontal and vertical positions of the inlet tube in the ion source were adjusted accordingly. After some experience was acquired in positioning the jet, the optimum conditions were readily reestablished each time the probe was inserted, and subsequent daily experiments required only minor tuning adjustments on background or solute ions. The API source did not require cleaning throughout a 5-day period of nearly continuous sample introduction through the DLI probe and inlet tube. The source itself operates at ambient temperature although the vaporization tube heated the source to 40-50 "C during the course of the day. The repeated introduction of relatively high levels (micrograms per minute) of the sulfa drugs did not result in increased backgrounds or in degraded source performance. This has been a previously noted feature of the atmospheric pressure source (16) which makes it attractive for applications in which raw, involatile (17),or relatively dirty samples are to be introduced, and results from the use of a carrier gas to transport sample vapors through the source with little chance of contacting a wall. Similarly,the continuous introduction of approximately 100 pL of solvent/min was readily accommodated without adversely affecting the source performance. The TAGA T M 6000 MS/MS contains an open central quadrupole region which is cryogenically pumped (15). The CID region in this quadrupole field is formed by a free jet expansion of the collision gas, ensuring that the fragmentation region is well defined and that no collisions occur in quadrupoles 1and 3. For these experiments, nitrogen was used as the collision gas, with an effective target thickness of approximately 2 X 1014molecules cm-2. Experiments were performed in several modes of operation. For LC/MS scans, quadrupole 1 (Q1) was set in the RF-only (total ion mode) and tuned to transmit ions of m / z > 100. The collision gas was turned off, and with Q1 and Q2 acting as high pass filters, Q3 was scanned over the desired mass range. The scan rate is controlled and determined by the intensity of the ion signal to ensure good statistical accuracy. The RF voltage applied to Q2 can be linked to either Q1 or Q3; for these experiments, Q2 was adjusted to 0.5 of Q3 voltage, providing good containment of both parents and daughters in the quadrupole field (15). Operation in the LC/MS/MS mode was performed by opening the valve to introduce the collision gas at a preset target thickness, setting the collision energy by adjusting the Q2 and Q3 rod offset voltages from the terminal keyboard, and switching Q1 to transmit only the parent ion of interest. The collision energy was adjusted to approximately 50 V for experiments with the sulfa drugs. This provided good fragmentation efficiency for all of the compounds tested, with at least one daughter ion intensity exceeding the intensity of the parent. For optimum sampling during the course of an LC peak during SIM experiments, mass spectrometer scanning was performed by rapidly jumping (under software control) Q3 from one daughter to the next, with up to eight ions being monitored in one run. Although Q1 could have also been switched continuously from one parent to another, these experiments were performed by switching from one parent to the next as each peak eluted, using the UV signal as a monitor.
RESULTS AND DISCUSSION The LC/MS mass spectrum of SM has been described previously (9). The abundant (M + 1)+ion at m / z 279 readily reveals the molecular weight of this sulfa drug but lacks any
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