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Note 64: Comparison of Various GC/MS Techniques For the Analysis of Black Pepper (Piper Nigrum)

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By David J. Manura and John J. Manura

Presented at PittCon 98, March 1998, New Orleans, LA

ABSTRACT
A large number of volatile and semi-volatile organic compounds are present in black pepper, which are responsible for its unique odor and flavor. These compounds include aromatics, hydrocarbons, terpenes and sesquiterpenes. In order to analyze and identify these compounds, a variety of extraction and sample introduction techniques are utilized for preparation of the sample for gas chromatographic separation of the analytes and for identification via a mass spectrometer.

The purpose of this paper is to study four different GC introduction techniques and a direct mass spec technique, including (1) solvent extraction, (2) direct probe mass spectrometry, (3) direct thermal extraction (DTE), (4) dynamic headspace and (5) solid phase micro extraction (SPME). Each technique has its own advantages and disadvantages. In this study, each technique was used to analyze a sample of black pepper to compare the ease and speed of each technique, the relative sensitivity of each method and the range of volatile and semi-volatile organic compounds extracted by each method. The purpose is to develop an understanding of each technique in order to aid the selection of an appropriate method for the analysis of other samples of volatile and semi-volatile organics.

Collectively, the five techniques identified more than 100 analytes in black pepper. However, the sample size required for each technique varied, as well as the range of light volatiles and semi-volatile organic compounds detected. The direct probe mass spec technique proved to be the least specific of the methods, identifying only piperine, the major alkaloid in black pepper. The greatest sensitivity with black pepper was achieved with the solvent extraction. Purge & Trap proved the least sensitive. The DTE had the shortest preparation time-over five minutes.

INTRODUCTION

A large number of volatile and semi-volatile organic compounds including aromatics, hydrocarbons, terpines and sesquiterpines are responsible for the unique odor and flavor of black pepper. There is a variety of extraction and GC sample introduction techniques for the separation and mass spectrometer identification of these analytes.

The purpose of this paper is to study four different GC/MS sample introduction techniques and one direct probe mass spec technique for the analysis of volatile and semi-volatile analytes present in black pepper. The techniques studied include: (1) solvent extraction, (2) dynamic headspace (P&T), (3) direct thermal extraction (DTE), (4) solid phase micro extraction (SPME) and (5) direct probe mass spectrometry. Each of these techniques has its own advantages and disadvantages. Therefore, each technique was evaluated in order to compare the equipment needed for analysis, speed of analysis, relative sensitivity, and range of volatile and semi-volatile organic compounds extracted. This study will help develop an understanding of each technique in order that chemists can select an appropriate method for the analysis and identification of the volatile and semi-volatile organics present in black pepper, as well as in other food samples.

EXPERIMENTAL

Materials

A sample of commercial black peppercorns was finely ground in a micro-mill for 5 minutes. This homogeneous mixture was used for all studies.

Gas Chromatograph/Mass Spectrometer Combination

The techniques (#1-4) utilized a HP 5890 Series II GC with Electronic Pressure Control, interfaced to a HP 5989 Mass Spectrometer. The GC injection port temperature was at 250 °C and the GC/MS transfer line was at 250 °C. A SGE BPX35, 0.25mm x 0.25µ x 60m GC capillary column was programmed with an initial temp of 30 °C for 5 min, a 6 °C min-1 ramp to 260 °C and a final temp of 260 °C for 5 min. Carrier gas flow was 0.90 ml per min of He. The HP 5989 MS operated in Electron Impact Mode (EI), with a mass range of 35 to 450 Daltons. The direct probe technique was conducted on a HP 5973 MSD with the SIS direct sample probe attachment.

Thermal Desorption System

The Scientific Instrument Services Short Path Thermal Desorption System (SPTD), Model TD3, was used for both the direct thermal extraction technique and the purge & trap techniques (techniques #2 and #3).

GC Micro Cryo Trap

A Scientific Instrument Services Model 951 GC Micro Cryo-Trap was placed at the front of the GC column in order to improve GC peak resolution. During the injection phase, the cryo-trap uses liquid CO2 to freeze the analytes at the front of the GC column at -70 °C. After injection was complete the cryo-trap was heated to 250 °C to release the analytes for analysis.

1. CHCl3 Solvent Extraction

Thirty (30.0) mg of black pepper was suspended in 4.0 ml of chloroform and shaken for 10 minutes. It was then filtered and the residue was extracted with an additional 4ml of chloroform. The combined filtrate was evaporated to 5.0 µL, and 0.5 µL was injected into the GC. This is equivalent to the analysis of 3.0 mg of black pepper.

2. Dynamic Headspace - Purge & Trap

The SIS Purge and Trap System was used in conjunction with the SPTD for the Purge & Trap technique. Black pepper (3.1 mg) and glass distilled water (5.0 mL) was put in a 25 mL P&T tube and connected to the Purge & Trap apparatus. The P&T tube was placed into a water bath maintained at 92 °C. Sample desorption tubes were packed with 150 mg Tenax® TA Resin for analyte trapping and attached to the P&T apparatus. The sample was purged with helium for 30 minutes at 40 ml per min and the analytes trapped on the desorption tube. An additional 40 ml per min dry purge was used during sample collection to eliminate the condensation of water on the adsorbent trap. After the P&T was complete, the desorption tube was purged with an additional 40 ml He to remove all water content. The Tenax desorption tube was then placed into the SPTD and thermally desorpted into the GC at 250 °C for 5 min. The analytes were cryo-focused at the front of the column and subsequently released and analyzed via the mass spectrometer.

3. Direct Thermal Extraction (DTE)

For the DTE technique, 3.2 mg of the pepper sample was placed on top of a glass wool plug in a 3.0 mm I.D. thermal desorption tube. The tube was purged with Helium for 20 sec, injected into the GC and thermally extracted for 5 min at 250 °C. The extracted analytes were cryo-focused at the front of the GC column. Afterward, the trapped volatiles were released and analyzed via the GC/MS system. Additional studies were conducted on the black pepper samples at several different extraction temperatures between 100 and 350 °C and also via a temperature programmed thermal extraction. These results were used to optimize the DTE system temperature parameters.

4. Solid Phase Micro Extraction (SPME)

A SuplecoTM Solid Phase Microextraction Assembly was used for the Solid Phase Microextraction (SPME) technique, with a 100 µm thick, Polydimethylsiloxane (PDMS) stationary phase. Black pepper (3.1 mg) was mixed with 5.0 mL water in a head space vial and capped. The mixture was heated to about 90 °C and allowed to sit for 30 min with the SPME fiber exposed to the liquid mixture in the sample vial. The SPME fiber was then injected into the GC injection port, thermally desorbed in the GC injection port for 5.00 minutes, and cryo-focused at the front of the GC column for subsequent analysis.

5. MS Direct Probe

For sample preparation, 2.6 mg of black pepper was suspended in 1.0 ml chloroform and shaken for 5 minutes. Then, 0.5 µL of solution was injected into the direct probe sample vial, the glass sample vial was inserted into the direct probe and the direct probe then inserted into the HP 5973 MSD. The direct probe was ballistically heated to 350 °C, and the mass spectrometer was scanned from 35 to 500 Daltons.

RESULTS

Utilizing the various techniques, more than 100 peaks were found in the GC chromatograms of the black pepper. Compounds identified via the HP ChemStation software included a large number of hydrocarbons, aromatics, terpenes and sesquiterpenes. These included piperine (the major aromatic compound present in black pepper), linalool, alpha- and beta-pinene, myrcene, 3-carene, limonene, and alpha- and beta-caryophyllene. These analytes identified were corroborated by the CRC Handbook of Medical Herbs. Each of the sample preparation and GC injection techniques was compared for its ability to detect these analytes, as well as its relative sensitivity. Figure 1 shows the relative sensitivity of the four GC/MS techniques. The total ion chromatograms have been adjusted for the same sample size and the same sensitivity scale.

Comparison of All Techniques
Figure 1.

Solvent Extraction

Solvent extraction has historically been the method of choice for the analysis of analytes in food samples. In this study the solvent extraction technique proved to be the most sensitive of the extraction methods. It also yielded the largest number of analytes (Figure 1). This technique does not discriminate based on analyte volatility, as the other techniques do. However, it can discriminate based on the solvent analyte solubility. This latter effect was not seen in this sample. In this technique the very light volatiles can be lost during sample preparation and sample concentration. This technique produced a large number of semi-volatile and non-volatile analytes that were not detected via the other techniques. Other disadvantages of this technique include the exposure of laboratory staff to the hazardous chloroform solvent and the cost of solvent disposal.

Dynamic Headspace

Purge & Trap was the least sensitive of the techniques studied (Figure 1). However, it did yield lower molecular weight (lower boiling point) analytes that were not detected in the other techniques (Figure 4). Static headspace techniques have also been used for the analysis of the very volatile analytes in black pepper and other samples. However, our previous studies have proven that static headspace is even less sensitive than dynamic headspace (P&T) and will not detect any of the higher boiling compounds or semi-volatile organics.

Direct Thermal Desorption at Various Temperatures
Figure 2.

Direct Thermal Extraction

The Direct Thermal Extraction technique is a preferred technique because it requires no sample preparation or extraction. The solid matrix sample is just placed directly into the thermal desorption tube and the analytes are thermally exacted from the sample. The DTE technique yielded a high abundance of volatile and semi-volatile organics, though slightly less than that achieved with solvent extraction (Figure 1). In a second study of this technique, several samples of black pepper were thermally extracted at temperatures from 100 to 350 °C (Figure 2). Higher molecular weight compounds did not begin to appear until the thermal extraction temperature was raised above 300 ºC. However, at this high temperature, some sample decomposition did occur. In a third study, the black pepper sample was analyzed via a temperature programmed thermal extraction from 100 to 250 °C (Figure 3). Utilizing the temperature programmed mode, it was expected that less sample decomposition would occur. In addition, several higher molecular weight analytes were detected, including a trace of peperine at 49.50 min.

Isothermic vs. Balistic DTE's
Figure 3.

SPME

SPME is a low-cost sample preparation technique that yielded fairly good results. However, the technique was less sensitive than the DTE and solvent extraction techniques (Figure 1). Historically, SPME has been less suitable for the analysis of the more volatile analytes; however, new fiber coatings have appeared recently that improve the detection of the low molecular weight analytes. The SPME technique detected a few peaks in the higher-mass range that were only detectable in the temperature programmed DTE and the solvent extraction techniques (Figure 4).

SPME vs. Purge & Trap
Figure 4.

Direct Probe

The direct probe technique is normally a poor choice for the analysis of complex mixtures of anlaytes in foods, because it does not offer the ability to separate or resolve the multiple analytes present in these samples. However, this technique can prove useful if a sample contains only one or two analytes that are significantly higher in concentration than other analytes in the sample. This technique has the advantage that it is quick (1 to 3 minutes). The literature reports that piperine is the major flavor component in black pepper. However, piperine was not detected in any of the other techniques, except the DTE temperature programmed technique, either due to piperine's high boiling point or solubility. The direct probe was the only technique that successfully identified piperene in black pepper (Figure 5). This is due to its high concentration relative to the other analytes present.

MS Direct Probe

Figure 5

CONCLUSION

No one anlytical method is optimal in all instances. There are a large number of factors that need to be considered in the selection of an anlaytical method. These factors include the physical properties of the sample, the sample matrix, the number of analytes of interest present in the sample, interfering compounds, the thermal stability of the sample, the cost and range of the equipment available, and the cost and time of the analysis.

Of the five techniques studied for black pepper, solvent extraction was the most sensitive technique, with the DTE technique the next most sensitive technique. The P&T technique was the least sensitive technique. The DTE and P&T techniques proved effective in the detection of the lowest boiling analytes. Static headspace is a good technique for the detection of the very volatile analytes. However, this technique cannot detect the less volatile and semi-volatile organics. The solvent extraction technique was the best method for the detection of the semi-volatile and non-volatile analytes. It was also the least costly in terms of additional hardware required. SPME was a good low-cost method for the analysis of many volatile and semi-volatile analytes, but it was less sensitive than the DTE and solvent extraction techniques. The MS direct probe technique was useful for the analysis of the piperine in black pepper and can also be used for the quick analysis of other samples.

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