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Note 50: The Analysis of Multiple Component Drug Samples Using a Direct Probe Interfaced to the HP 5973 MSD

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By John J. Manura
1999

INTRODUCTION

Direct probe sample introduction to a mass spectrometer has been widely used to analyze single component samples or samples with one or two major analytes. In the past, multiple component samples were difficult if not impossible to interpret using the direct probe sample introduction when the mass spectrometer is used in the electron impact (EI) mode. The forensic community has widely used the mass spectrometer direct probe sample introduction with the mass spectrometer operating in the chemical ionization (CI) mode (1-5). The CI technique produces simplified mass spectra to one or two ions for each analyte, permitting the interpretation of samples with up to 10 analytes present in a single sample. However, the CI technique is not a definitive identification method and the absolute identification of drug samples must be determined via other analytical techniques. To obtain a positive identification of a drug sample, the mass spec should be operated in the EI mode. This has previously not been possible with multiple component samples using the direct probe introduction technique.

Figure # 1 - HP Probe and Probe Inlet System

It is now possible to analyze multiple component samples via the direct probe using the new high sensitivity Hewlett Packard 5973 MSD interfaced with the SIS Direct Probe sample introduction system. This can be accomplished in the EI mode with the use of the H.P. ChemStation Software that is normally used for GC analysis. This software permits the user to analyze multiple components in a sample analyzed via the direct probe mass spectra run by performing the following refinement of the mass spectral data:

1. Analyze the data using the selected ion extraction module to determine the mass spec scans with the highest concentration of the analytes of interest.

2. Analyze the data by averaging multiple mass spec scans across the area of the greatest ion intensity for the analyte of interest.

3. Permit background subtraction from either the front or back side of the data analyzed in step 2 above. This cleans up the mass spectra by removing interferences and background peaks from the sample mass spectra.

The purpose of this paper is to demonstrate the analysis of a multiple component drug sample using the SIS heated direct probe interfaced to the Hewlett Packard 5973 MSD. An over the counter antihistamine preparation containing Phenylpropanolamine, Brompheniramine Maleate and a variety of other binding materials and diluents was used for this analysis. The heated direct probe will permit the thermal separation of the various analytes in the sample which can then be positively identified from the mass spectral data.

Experimental

The SIS Direct Probe Inlet and Probe (Figure 1) was attached to the GC/MS interface port on a H.P. 5973 MSD. The mass spec probe was 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 1.7mm diameter by 12.75 mm long into which the sample is inserted. The sample vial is then inserted into the tip of the probe where it is held in place by a small spring.

Each 600 mg tablet contains the following major active ingredients. 

     Brompheniramine maleate:   12 mg
     Phenylpropanolamine:       75 mg
The remainder of the tablet consists of acacia, acetylated monoglycerides, calcium sulfate, carnauba wax, citric acid, edible ink. FD&C Blue 1, Gelatin, hydrogenated castor oil, magnesium stearate, magnesium trisilicate, pharmaceutical glaze, polysorbates, povidone, silicon dioxide, steraryl alcohol, sucrose, titanium dioxide, white wax and wheat flour.

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 the amount of drugs present in the chloroform solution assuming 100% extraction into the chloroform solution. This chloroform solution should therefore contain a maximum concentration of the two active ingredients as follows: 

   Phenylpropanolamine    140 ng/ul
   Brompheniramine         23 ng/ul
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.

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 250 and the quads to 150 degrees C. The sample 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. The total analysis time was 1.0 minute.

After the sample analysis was complete, the total ion chromatogram was viewed using the ChemStation data analysis software and the sample was then further analyzed using the selected ion extraction function of the Chem Station software searching for the 44 ion which is the base ion for phenylpropanolamine and the 249 ion which is the base ion for bromopheniramine. Additional mass ions were evaluated to determine the presence of other analytes as well as interfering compounds in the sample.

Results

Figure # 2 - Direct Probe Analysis of Drug Mixture

The total ion chromatogram for the 1.0 minute probe analysis time is shown in Figure 2A. It consists of one major peak at 0.5 minutes and several other small humps in the total ion chromatogram. 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 data was first analyzed by extracting the major ion for Phenylpropanol-amine (mass 44) and the major ion for Brompheniramine (249) using the selected ion extraction method in the ChemStation data analysis package. This produced the two chromatograms shown in Figure 2C and Figure 2E. 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 2B. When the mass spectrum for the Brompheniramine peak (Figure 2E) was analyzed, it was found to contain a great deal of extraneous ions including 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 was determined and is displayed in Figure # 2D.

Figure # 3 - Mass Spectra of Drug Mixture

In Figure 3 are displayed the mass spectra of each of the four selected ion peaks determined in Figure 2. The first spectra (Figure 3B) 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).

The second spectra (Figure 3C) 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 2D and 2E were used to obtain a clean mass spectra of the Brompheniramine. As can be seen from Figure 2E, the Brompheniramine is eluted slightly before the larger interfering peak shown in Figure 2D. 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 3E 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 3D 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. The mass spectrum consists of major ions at 55, 69, 83, 97, and 111and minor peaks at 252, 224, 196 and many others. 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.

Conclusion

The analysis of multiple analyte 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 analysis can be performed in only 1.0 minute. The total ion chromatograph consists of broad and often overlapping peaks for each of the analytes. The Chem Station 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 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 1.5 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. The direct probe introduction system interfaced to the HP 5973 will be a valuable technique for the analysis of many drug and pharmaceutical samples, where fast sample analysis is required.

References

(1) Identification of Drugs by chemical Ionization Mass Spectroscopy, Part II, Richard Saferstein, Jew Ming Chao and John Manura, J. of Forensic Science, Vol 19, No.3, 1974, pp 463-485.

(2) Identification of Heroin and its Diluents by Chemical Ionization Mass Spectroscopy, Jew Ming Chao, Richard Saferstein and John Manura, Analytical Chemistry, Vol 46, No. 2, Feb. 1974, pp 296 - 298.

(3) Drug Detection in Urine by Chemical Ionization Mass Spectroscopy, Richard Saferstein, John Manura. and P.K. De, J. of Forensic Science, Vol 23, No. 1, 1978, pp 29 - 36.

(4) Drug Detection in Urine by Chemical Ionization Mass Spectroscopy, Part II, Richard Saferstein, John Manura, Thomas Brettell and P.K.De, J. of Analytical Toxicology, Vol. 2, Nov-Dec 1978, pp 245 - 249.

(5) Chemical Ionization Mass Spectrometry of Morphine Derivatives, Richard Saferstein, John Manura and Thomas Brettell, J. of Forensic Science, Vol 24, 1979, pp 312 - 316.