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Note 48: Demonstration of Sensitivity Levels For the Detection of Caffeine Using a New Direct Probe and Inlet for the HP 5973 MSD

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By John J. Manura
1999

INTRODUCTION

The direct probe has routinely been used for mass spectrometer sample analysis for over 30 years. The technique has the advantage of being very fast and permits the analysis or screening of a large number of samples in a normal work day. It permits the analysis of many samples which can not be chromatographed or analyzed by other techniques. However, the technique can not separate or resolve more than two or three analytes in a sample. Samples must be relatively clean or the analyte of interest must be the major component in the sample. Sample clean up can often be accomplished by a quick solvent extraction or the use of a solid phase extraction cartridge. The direct probe technique has sensitivity limitations due to the lack of resolution of the sample analytes as compared to GC sample analysis with very narrow peak widths for each of the analytes. However, the short analysis times of the direct probe technique make it the method of choice for drug screening and quality control sample analysis in the forensic and pharmaceutical industry.

Samples are normally introduced into the HP MSD instruments through a gas chromatograph via the GC/MS heated interface on the right side of the MSD. A direct probe was not available for the 5971 and 5972 instruments. This was due to the design of the MSD instruments and the use of the GC/MS transfer line to indirectly heat the MSD source. The new Hewlet Packard 5973 MSD utilizes a cartridge heater and heater block to directly heat the mass spec source. This now permits the use of a direct probe. SIS has designed and built a direct probe and probe inlet system for the HP 5973 (Figure 1). This system mounts onto the GC/MS transfer line port and uses an indexed probe introduction guide to permit the introduction of a direct (solids) probe into the MSD source.

Figure # 1 - Components of the HP 5973 Probe Inlet System

The purpose of this study is to demonstrate the use of the new HP 5973 direct probe and probe inlet system. Analysis of a caffeine standard sample illustrates the use of the direct heated probe to determine the levels of sensitivity which can be achieved with this technique.

Experimental

Figure # 2 - HP Probe Inlet System Mounted On the HP 5973 MSD

The SIS Direct Probe Inlet and Probe (Figure 1), is designed to mount on the H.P. 5973 MSD. In order to install the system, it is necessary to first remove the gas chromatograph and the heated GC/MS interface from the MSD. The probe inlet system then attaches to the side of the MSD using the same three mounting holes which held the GC/MS interface onto the side of the MSD (Figure # 2). The probe inlet system (Figure 1) consists of a ¼ turn ball isolation valve to permit the introduction of the probe into the mass spec, a ¼ turn rough pumping valve attached to an Edwards 1.5 rough pump, a dual PTFE sealing system to provide long life vacuum seals to the probe, and an indexed dual guide rod introduction system. This guide rod system is indexed at three positions to permit the easy introduction of the probe into the vacuum system with automatic stops at the first seal position, the second seal position and 1 cm distance from the source (cooling position). These automatic stops enable the accurate positioning of the probe, eliminates scoring of the probe rod due to closing the ball valve on the probe rod and minimizes the accidental venting of the mass spec by inadvertently pulling the probe out past the vacuum seals before closing the isolation valve. The mass spec probe can be heated ballistically up to 450 degrees C at a ramp rate in excess of 500 degrees per minute. The probe can also be temperature programmed for slower heating applications. The probe shaft is only ¼" in diameter and the probe handle is 1.25" in diameter. This provides for ease of handling during sample introduction. The probe uses flared glass sample vials 1.7mm diameter by 12.75 mm long for sample containment. The sample vial is then inserted into the tip of the probe where it is held in place by a small spring.

Pure anhydrous Caffeine (Sigma Chemical Company, St. Louis, MO) was used for the following analysis via the direct probe technique. A solution of caffeine was prepared at a concentration of 10 mg/liter (10 ng/ul) in Methanol. This solution was injected into the direct probe sample vials using a GC syringe to prepare sample vials with the following amounts of caffeine:.

Sample    Sample Volume    Amount of Caffeine
  1           0.5 ul             5.0 ng
  2           1.0 ul            10.0 ng
  3           2.0 ul            20.0 ng
  4           3.0 ul            30.0 ng
After sample injection, the methanol solvent in each of the sample vials was allowed to evaporate to dryness before analyzing any of the samples. This was accomplished in about 15 minutes by heating the sample vials in a sample vial block to 60 degrees C.

Figure # 3 - Direct Probe Analysis of 10 ng Cafeine

The HP 5973 MSD was operated in the EI mode and was scanned from 50 to 200 daltons at 1 scan per second. The mass spec source temperature was set at 250 and the quads at 150 degrees C. Each direct probe sample was inserted into the mass spec probe and inserted through the vacuum lock system of the probe inlet into the mass spec source of the HP 5973 MSD. Each sample was then heated ballistically to 250 degrees (at > 500 degrees per minute), and the mass spec was scanned to analyze the thermally extracted sample. The total analysis time was 1.5 minutes per sample. After the sample analysis was complete, the total ion chromatogram (Figure 3 - top) was viewed using the ChemStation data analysis module and then the sample was then further analyzed using the selected ion extraction function of the ChemStation software for the 194 ion of caffeine (Figure 3 - bottom). The average mass spectrum was then taken over a 0.10 minute time range at the highest intensity range of the resulting peak and the background from the front of this broad peak was subracted.

Results

The total ion chromatogram for a 10 ng sample of Caffeine is shown in the top drawing of Figure 3. The selected ion chromatogram for mass 194 was then determined utilizing the ChemStation software (bottom drawing of Figure 3). The result was a near gaussean shaped peak with tailing on the back side an a peak half width of about 0.40 minutes. The 194 ions are part of the total ions present in the sample as shown in the top drawing of Figure 3. The other ions consisted of other background materials including several hydrocarbons. This use of the selected ion program in the ChemStation software is a very useful tool to search for a particular analyte in a direct probe sample, and will enable the user to accurately analyze the most intense section of the total mass scans and also select an area from which to subtract the background. The use of background subtraction is quite useful in order to clean up a mass spectrum for more accurate sample identification using standard mass spec libraries.

Figure # 4 - Direct Probe Analysis of Caffeine Sensitivity Study (Selected Ion Extraction - 194)

The results of the selected ion chromatograms for each of the 4 sample sizes of the caffeine samples analyzed are shown in Figure 4. Sample sizes down to 5.0 ng were easily detected and identified using the direct probe technique. However, at the lower concentrations, the signal became quite weak and the signal to noise ratio decreased. The mass spectrum was taken by analyzing the average of a 0.10 minute time at the top of each peak in Figure 4 and the background was subtracted from the front of each peak utilizing the ChemStation software. The results are shown in Figure 5. Caffeine has a molecular weight of 194 and produces a molecular ion at mass 194. The minor ions at 109, 82, 67, 55, 137 and 165 also originate from the caffeine. Although the Caffeine is easily detected in all the samples, it is less obvious in the 5.0 ng sample due to the hydrocarbon background. Detection of caffeine below 5.0 ng would be more difficult but could be achieved by scanning a narrow mass range (i.e 150 to 200 daltons) or by using the mass spec in the selected ion mode (SIM).

Figure # 5 - Mass Spectra of Caffeine via Direct Probe Sensitivity Study (50 to 200 daltons)

Additional studies have recently been done, operating the mass spec source at 350 degrees C and maximizing the mass spec scan rate for optimum sensitivity. As a result, we have been able to analyze caffeine samples down to 1 nanogram size.

Conclusion

The new SIS direct probe and probe inlet system has proved to be a useful addition to the new Hewlet Packard 5973 MSD. The probe inlet system permits the direct introduction of the probe through what is normally the GC/MS interface line on the MSD. The indexed probe introduction system permits the easy insertion of the probe without the chance of inadvertently venting of the mass spec or scoring of the mass spec probe shaft. Samples were analyzed in less than 1.5 minutes with this technique. The direct probe analysis technique has proved to be a very sensitive technique for solid sample analysis with detection levels of 5 to 10 ng of Caffeine injected. The broad peak width (0.4 minute peak half width) is quite large as compared to a GC analysis and contributes to the loss in sensitivity as compared to conventional GC analysis where peak widths are normally less than 5 seconds. However, the outstanding sensitivity of the new HP 5973 MSD enables the direct probe analysis of sample levels comparable to many GC samples analyzed via the older 5971 MSD instruments. The direct probe introduction system interfaced to the HP 5973 will be a valuable technique for the analysis of many samples, where fast sample analysis is required such as in drug screening, for the quality control analysis of samples and for the analysis of samples which can not be chromatographed.