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Note 58: Direct Probe Analysis and Identification of Multicomponent Pharmaceutical Samples via Electron Impact MS

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By John J. Manura and Steven Colby

Presented at ASMS Meeting, Palm Springs, CA., June 5, 1997

INTRODUCTION

The Direct Probe has been used for many years to introduce samples into the mass spectrometer. This technique has the advantage of quick analysis times with minimal sample preparation. However, it has the disadvantage in that it could only be used for the analysis of relatively pure samples due to the complexity of the mass spectrum of multiple component samples. This has been overcome by analyzing direct probe samples using the Chemical Ionization mass spec technique. Chemical Ionization simplifies the mass spectrum of each analyte to a few ions enabling the identification of multiple analytes in a single sample. However, these simplified spectra are not a conclusive identification of the analytes.

A new microprocessor and computer-controlled direct probe has been developed for the bench top mass spectrometer to permit both controlled temperature ramping as well as ballistic heating of the mass spectrometer direct probe. Through the use of rapid ballistic heating of the direct probe at rates greater than 500 degrees per minute, it is possible to separate multiple component samples while utilizing the mass spectrometer in the Electron Impact mode. The thermal separation achieved with this technique can best be compared to the results that would be obtained on a short packed GC column. The resulting peaks are broad (0.1 to 0.2 minutes) and are often overlapping. Utilizing the new PC sample analysis software and the use of background subtraction techniques, the multiple component mixtures can be easily analyzed and positively identified using the standard PC library search techniques.

The purpose of this paper is to demonstrate the analysis of a multiple component drug sample using the heated direct probe interfaced to the Hewlett Packard 5973 MSD. An extracted drug sample and a powdered drug sample are analyzed to demonstrate the application of this technique for the identification of multiple component samples.

Experimental

The SIS Direct Probe Inlet and Probe was attached to the GC/MS interface port on a H.P. 5973 MSD. The mass spec probe can be heated ballistically 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 uses flared glass sample vials into which the sample is inserted.

Sample 1

The first sample analyzed was an over the counter pharmaceutical. Each 600 mg tablet contains Brompheniramine maleate (12 mg) and Phenylpropanolamine (75 mg). The sample was prepared by breaking the tablet and weighing 11.4 milligrams of the tablet into a flask containing 10 ml of chloroform plus 10 ml of 1.0 Normal NaOH. The mixture was thoroughly shaken to dissolve the drug powder and extract the basic drugs into the chloroform solution. Calculations were performed to determine that the chloroform solution should contain a maximum concentration of 140 ng/ul Phenylpropanolamine and 23 ng/ul Brompheniramine. Two (2.0) ul of the extracted chloroform solution was injected into a direct probe sample vial and the solvent was allowed to evaporate to dryness. This was accomplished in about 15 minutes by heating the sample vial in a sample vial block to 60 degrees C.

Sample 2

The second sample was a pharmaceutical containing Acetominophen, Caffeine and Aspirin. This sample was not extracted. Instead the tablet was crushed, and a small amount (<100 ug) of the powder was placed inside the direct probe capillary. This was accomplished by first inserting some of the powder into the sample vial and then shaking the powder out until only a trace residue of the powder remained.

The HP 5973 MSD was operated in the EI mode and was scanned from mass 40 to 350 daltons at 1 scan per second. The mass spec source temperature was set to 350 degrees C and the quads to 150 degrees C. The sample vial was then 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. The sample was then heated ballistically to 300 degrees, and the mass spec was scanned continuously to analyzed the thermally extracted analytes. Sample 2 was temperature programmed at 200 degrees per minute from 30 to 350 degrees C.

After the sample analysis was complete, the total ion chromatogram was viewed using the ChemStation data analysis software. The sample was then further analyzed using the selected ion extraction function of the ChemStation software for ions of major intensity in the analytes suspected to be in the samples.

Results

Sample 1

The total ion chromatogram for extracted pharmaceutical drug mixture is shown in Figure 1. It consists of one major peak at 0.5 minutes and several other small humps. The resulting chromatogram can be equated to a packed GC column run with poor resolution. However, with the software tools available in the ChemStation software, a great deal of information can be extracted from this data. The PC software was used to extract the major ion for Phenylpropanol-amine (mass 44) (Figure 1C) and the major ion for Brompheniramine (249) (Figure 1E) using the selected ion extraction method. In addition, the small peak at 0.24 minutes was determined to consist of a major ion at mass 281 which was analyzed via the selected ion extraction method and displayed in Figure 1B. When the mass spectrum for the Brompheniramine peak (Figure 2E) was analyzed, it was found to contain additional ions at 69, 83 and 97. These were assumed to be from an interfering compound which was thermally extracted at the same time interval as the Brompheniramine. Therefore the selected ion extraction for the 97 ion is displayed in Figure 1D.

Figure 1

Figure 1 - Sample 1 - Direct Probe Analysis of Extracted Pharmaceutical Sample Containing Phenylpropanolamine and Brompheniramine.

In Figure 2 are displayed the mass spectra of each of the four selected ion peaks determined in Figure 1. The first spectra (Figure 2B) is the unknown peak at about 2.4 minutes which contains a major ion at 281. This spectra was obtained by averaging the spectra over the time interval 0.233 to 0.240 minutes and subtracting the background spectra at 0.20 minutes. A library search of the Wiley mass spec data base identified this peak as octamethylcyclotetrasiloxane (a common silicone).

Figure 2

Figure 2 - Sample 1 - Mass spectra Data For Extracted Pharmaceutical Sample

The second spectra (Figure 2C) was obtained by averaging the mass spectra in the time interval 0.374 to 0.378 minutes and subtracting the background spectra a 0.26 minutes. It contained a major ion at mass 44 and minor ions at 51, 77, 79 and 91 which are identical to the spectra peaks in Phenylpropanolamine.

The selected ion chromatograms displayed in Figures 1D and 1E were used to obtain a clean mass spectra of the Brompheniramine. As can be seen from Figure 1E, the Brompheniramine is eluted slightly before the larger interfering peak shown in Figure 1D. Therefore, by averaging the spectra over the time interval 0.471 to 0.500 and subtracting the background at 0.56 minutes, the mass spectrum shown in Figure 2E was obtained. The spectra is clean of background peaks and contains two major ions of equal intensity at 247 and 249 (indicating the presence of a Bromide ion). The additional minor ions at masses 42, 44, 58, 72, 115, 167 and 180 confirm the identification of Brompheniramine. Without the background subtraction capabilities of the ChemStation software, the clean mass spectra of this analyte would have been impossible.

The final mass spectrum shown in Figure 2D is the interfering compound. It was obtained by averaging the mass spectra over the time range 0.534 to 0.537 minutes and subtracting the background due to the Brompheniramine at 0.50 minutes. This compound was identified as 1-octadecanol by the Wiley NBS Library..

The above data analysis demonstrates the use of the selected ion extraction and background subtraction software functions in the ChemStation software to analyze multiple analytes in a drug sample. At least 4 separate analytes were resolved and the mass spectra of each obtained for positive identification. This sample was analyzed in only 1.0 minute.

Sample 2

The total ion chromatogram for solid pharmaceutical drug mixture is shown in Figure 3A. It consists of one major peak at 1.1 minutes and several other humps in the total ion chromatogram. The PC software was used to extract the major ions for Acetominophen (mass 151), Caffeine (194) and Aspirin (120) using the selected ion extraction method. This produced the chromatograms shown in Figure 3B, 3C and 3D.

Figure 3

Figure 3 - Sample 2 - Direct Probe Analysis of Solid Pharmaceutical Sample Containing Acetominophen, Caffeine and Aspirin

In Figure 4 are displayed the mass spectra of each of the three selected ion peaks detected in Figure 3. Each of the peaks was analyzed by selecting the mass spectra at the area of highest ion sensitivity and then subtracting an area near the base of the compound to remove any interfering analytes. As a result, all three of the analytes were detected and positively identified utilizing the PC software and library search methods.

Figure 4

Figure 4 - Sample 2 - Mass Spectra Data For Solid Pharmaceutical Sample

This sample is unique because it demonstrates the analysis of multiple component drug mixtures without the need for any sample preparation or solvent extraction. Both basic and acidic pharmaceuticals were identified in a single sample analysis in 1.5 minutes.

Conclusion

The analysis of multiple component mixtures of pharmaceutical or forensic drug samples can be readily analyzed using the HP 5973 MSD in the EI mode with sample introduction via a heated direct probe. This is possible due the rapid direct probe ramp rates that permit the detection of small sample sizes (down to 5 ng). Little or no sample preparation is required for this analysis. If is often possible to analyze solid samples directly by placing the powder directly into the mass spec probe. The resulting total ion chromatograph consists of broad and often overlapping peaks for each of the analytes. However, PC software functions including selected ion extraction, spectra averaging and background subtraction can all be used to clean up the mass spectra to produce library searchable and identifiable mass spectra for each of the analytes.

The heated direct probe and sample introduction system has proved to be a useful addition to the new Hewlett Packard 5973 MSD for the analysis of drugs and pharmaceuticals. The probe inlet system permits the direct introduction of the probe through what is normally the GC/MS interface line on the MSD. Samples are normally analyzed in less than 1.0 minutes using the ballistic heating probe method. However, the probe can be temperature programmed at slower rates for the purpose of improving the separation of mixtures of analytes in a single sample. The highly sensitive HP 5973 MSD and the versatile ChemStation software permit the analysis of pure and mixed drug samples via the EI mass spec probe technique to achieve a fast and definitive method of analysis. This technique is a valuable technique for the analysis of many drug and pharmaceutical samples, where fast sample analysis is required. Applications include quality control and drug screening.