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Chromatographia

, Volume 82, Issue 1, pp 317–323 | Cite as

Determination of Volatile Amines Using Needle-Type Extraction Coupled with Gas Chromatography–Barrier Discharge Ionization Detection

  • Ikuo UetaEmail author
  • Yohei Nakamura
  • Hiroto Fujikawa
  • Koji Fujimura
  • Yoshihiro Saito
Original
  • 110 Downloads
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Abstract

Volatile amines including ammonia, methylamine, dimethylamine, ethylamine, and trimethylamine were extracted using a needle-type extraction device followed by determination using a gas chromatography–barrier discharge ionization detector. The analyte amines were extracted based on their reaction with an organic acid in the needle packed by the adsorbent. The extracted compounds were desorbed by heating the extraction needle in the GC injection port and introduced into a GC column. After optimization of the desorption process, the proposed method was quantitatively evaluated for linearity, repeatability, and sensitivity. The limits of detection for ammonia, methylamine, dimethylamine, ethylamine, and trimethylamine with an air sampling volume of 100 mL were 0.03, 0.1, 1, 0.06 and 0.05 µg L−1, respectively. The method was applied to the determination of amines from raw fish and raw meat samples.

Keywords

Volatile amine Ammonia Needle extraction Sample extraction Gas chromatography–barrier discharge ionization detector 

Introduction

Low-molecular-weight amines are used in many fields, including as precursors to dyes, pesticides, and pharmaceuticals. These amines tend to have unpleasant odors, which are often described as “fishy,” and are known to be indicators of fish and meat spoilage. Exposure to higher concentrations of amines for long durations can have many adverse effects on humans, including nose and throat irritation and dyspnea [1, 2].

Typical methodologies for the determination of gaseous ammonia involve collection in a borate buffer solution followed by spectrophotometric detection as the indophenol [3] or ion chromatographic analysis [4, 5]. Gas chromatography (GC) has also been employed for the analysis of volatile amines, where flame ionization detection (FID) or mass spectrometry (MS) are used as to detect the analytes [6, 7]. However, FID shows no response to non-carbon compounds such as ammonia, and detection of ammonia by typical GC–MS instruments is quite difficult because of its low molecular weight. Derivatization of amines has been used to reduce the polarity and to improve the sensitivity in conventional GC detectors [8]. Therefore, simultaneous and sensitive determination of ammonia and other amines is somewhat difficult when using these conventional GC detection methods.

Recently, GC detectors that use helium plasma, termed barrier ionization detectors (BIDs), have been developed. BIDs can detect almost all volatile compounds except helium and neon, and they exhibit a greater sensitivity than FID [9]. BIDs clearly show particular advantages in the sensitive detection of compounds with low molecular weights that are poorly detectable. If in the case of no response is obtained by FID, such as for the detection of carbon dioxide, formic acid, and ammonia, the advantages are quite significant.

Several sample preparation methods have been employed for the sensitive determination of trace amines by GC. Among them, solid-phase microextraction (SPME) has recently been utilized to successfully extract volatile amines from gaseous, aqueous, and food samples [10, 11, 12]. In addition, on-fiber-derivatization SPME also provides sensitive detection of such analytes [13]. Furthermore, needle-type extraction (or needle-trap extraction) devices have recently emerged as suitable sample preparation tools for volatile organic compounds (VOCs) in GC analysis [14, 15, 16, 17]. These devices are typically constructed with a particular adsorbent packed into a stainless-steel needle, and they are designed for the active sampling of gaseous samples [18, 19, 20, 21]. Active sampling is typically more suitable for the extraction of volatile compounds than static sampling. Therefore, needle-type extraction is expected to be more rapid and sensitive for the determination of volatile amines [22].

Previously, our research group has reported the determination of formic and acetic acids in gaseous [23] and aqueous [24] samples with GC–BID using a needle-type extraction device. By combining needle extraction with GC–BID, these analytes were rapidly and sensitively determined.

This manuscript is the first report about determination of volatile amines with preconcentration by needle-type extraction followed by GC–BID analysis. After optimization of the analytical conditions, analytical parameters such as sensitivity, linearity, and repeatability were evaluated. Finally, the method was applied to the determination of volatile amines in raw fish and meats samples.

Experimental

Chemicals

Ammonia (28% in water), methylamine (MA, 40% in water), and dimethylamine (DMA, 50% in water) were obtained from Tokyo Kasei Kogyo (Tokyo, Japan). Ethylamine (EA, 70% in water) was purchased from Wako Pure Chemical Industries (Osaka, Japan). Trimethylamine (TMA, 30% in water) was obtained from Junsei Chemical Co., Ltd. (Tokyo, Japan).

Preparation of Standard Gas Samples

An appropriate amount (typically a few microliters) of an analyte solution was introduced into a 1.0 L vacuum glass vessel and evaporated therein. After suppling 1.0 L of pure N2 gas to the glass vessel, a few milliliters of the prepared standard gas were collected using a gas-tight syringe and injected into a gas sampling bag (Smart Bag PA, GL Sciences, Tokyo, Japan) containing an appropriate volume of pure N2 or air. The gas sampling bag was thoroughly purged with purified N2 before use. To obtain standard gas samples with lower concentrations, the above standard gas was further diluted by a similar process using another gas sampling bag. All the standard gas samples were prepared at room temperature (ca 25 °C).

Needle-Type Extraction Device

A custom-made extraction needle was prepared by Shinwa Chemical Industries (Kyoto, Japan). The needle was prepared by packing adsorbent particles into a stainless-steel needle (85 × 0.5 mm I.D., 0.7 mm O.D) with a tip hole. As the adsorbent, non-volatile carboxylic acid coated macroporous terephthalic acid particles (60/80 mesh) was used. The analyte amines react with the acid and are collected on the adsorbent (Fig. 1). The packing length of the sorbents was 25 mm. A small amount of Zylon filament (Toyobo, Shiga, Japan) was also placed at each end of the packed section to fix the sorbent in place. After packing of the adsorbent and Zylon filaments into the needle, a luer lock connector was connected.

Fig. 1

Illustration of the extraction needle and the analytical procedure used for determining gaseous samples

GC Measurement

A Shimadzu GC-2010 Plus capillary GC–BID instrument (Tracera, Shimadzu Corporation, Kyoto, Japan) was used for all GC measurements. Separation was conducted using a CP-Volamine capillary column (30 m × 0.32 mm, Agilent Technologies, Santa Clara, CA, USA). The column head pressure and split ratio were set at 150 kPa and 10:1, respectively. The column temperature was maintained at 35 °C. The injector temperature and the detector temperature were set at 240 and 250 °C, respectively. A base-deactivated liner (Restek Corporation, PA, USA) of 3.5 mm i.d. was used as the inlet liner.

Sampling and Desorption Methods

A vacuum sampling device (Kitagawa AP-20, Komyo Rikagaku Kogyo, Tokyo, Japan) was used to collect gas samples. The sampling time was effected by particle size and packing amount of the adsorbent, and in this study approximately 10 min for a sampling volume of 100 mL. After the extraction of the analytes, the extraction needle was attached to a gas-tight syringe, and then 0.3 mL of N2 gas was collected via the needle as the desorption gas. The extraction needle was then inserted into the heated GC injection port. The extracted analytes were thermally desorbed from the adsorbent as the respective amines and introduced into the separation column. The desorption of the analytes by nitrogen gas was performed for 3 s. A scheme for the extraction and desorption procedures is given in Fig. 1. For real sample analysis, after the sample collection, 50 mL of pure N2 gas was collected through the extraction needle using the vacuum sampling device to remove excess water from the extraction sorbents.

Results and Discussion

Optimization of the Method

The desorption temperature (i.e., injector temperature) significantly effects the desorption rate of the analytes extracted in the needle [20, 23]. Therefore, the desorption temperature was initially optimized using a standard gas mixture. The desorption of the analytes was assessed based on a comparison of the peak area obtained for the first desorption with the total peak area obtained for the first and second desorptions. Figure 2 shows the desorption rates of the analytes at desorption temperatures ranging from 200 to 250 °C. The desorption rate of TMA increases with desorption temperature, while the other amines are completely desorbed at all the investigated desorption temperatures. Higher temperatures showed decreased lifetime of the adsorbent which could be due to sublimation of organic acid. Therefore, the desorption temperature was set at 240 °C. The extraction efficiency gradually decreases upon repeated use due to degradation of the adsorbent. However, the extraction needle can be reused more than 50 times at this injection temperature where decrease of analyte peak areas were within 10%.

Fig. 2

Optimization of the desorption temperature. Sample concentrations: ammonia 10 µg L−1; MA, EA, and TMA 30 µg L−1; DMA 300 µg L−1

The relationship between the gas sampling volume and peak area was investigated, as illustrated in Fig. 3. Each peak area was calculated based on the ratio of the peak area obtained with a sampling volume of 100 mL. Except for that of TMA, the analyte responses increase with increasing sampling volume. The TMA peak area reaches a plateau at a sampling volume of 200 mL. This could be due to the lower reactivity between TMA and the organic acid used as the adsorbent. As shown in Fig. 4, higher concentrations of TMA result in a plateau in the peak area at lower sampling volumes. This clearly indicates that the linear range for TMA detection is largely dependent on its concentration.

Fig. 3

Relationship between sampling volume and peak area of amines. Sample concentrations: ammonia 500 µg L−1; MA 4000 µg L−1; DMA 4000 µg L−1; EA 500 µg L−1; TMA 500 µg L−1

Fig. 4

Peak area for TMA at different concentrations and sampling volumes

Evaluation of the Method

The limit of detection (LOD) and the limit of quantification (LOQ) were defined as signal–noise ratios (S/N) of 3 and 10, respectively. The LODs and LOQs for the proposed method using standard gas samples at a sampling volume of 100 mL are summarized in Table 1. In the present analytical method, EA is not completely separated from DMA and TMA (Fig. 5). Consequently, EA was measured alone. The lower response for DMA than that for the other investigated amines was also confirmed by injection of a higher-concentration standard gas into the GC–BID instrument without preconcentration. The upper LOQs for TMA were determined to be more than 3 µg L−1, and others were more than 50 µg L−1 with the determination coefficients greater than 0.99. The analyte sensitivity is effected by the humidity of the gaseous sample because the reaction efficiency of the organic acid on the sorbent changes with humidity. More basic compounds, such as ammonia, are more affected by the humidity, and a humid air sample results in higher detector responses than those for dry samples. Therefore, to quantify ammonia in humid gaseous sample, using standard gas that adjusted a desired relative humidity [25, 26] or standard addition method could be needed. However, the TMA response is not effected by the humidity. The repeatability of the method was confirmed by calculating the relative standard deviation (RSD) of the peak area of a standard sample (n = 5). The RSDs for all the analytes are lower than 12%.

Table 1

LODs and LOQs for standard volatile amines

 

Ammonia

MA

DMA

EA

TMA

LOD (µg L−1)

0.03

0.1

1

0.06

0.05

LOQ (µg L−1)

0.09

0.3

3

0.2

0.15

Sampling volume: 100 mL

Fig. 5

Typical chromatogram for the determination of standard amine samples. Sample concentrations: ammonia 1 µg L−1; MA, EA and TMA 5 µg L−1; DMA 30 µg L−1. Sampling volume: 100 mL

The storage recoveries of the extracted analyte amines from the extraction needle were investigated for storage at room temperature (25 °C) and in a refrigerator (4 °C). To calculate the extraction recoveries, the peak areas for each standard amine extracted with the needle were measured. Then, the same gaseous sample was extracted with the same extraction needle again and stored. After extraction, both ends of the extraction needle were capped with PFTE plugs, and the needle was stored at a constant temperature. The stored analyte was then injected into the GC instrument and the storage recovery was calculated based on the ratio of the peak area obtained for the stored sample to that of the non-storage sample. The storage recoveries are summarized in Table 2. All the analytes are successfully stored for a period of 6 h, although the recoveries significantly decrease at a storage period of 24 h. The storage recoveries slightly improve for the storage at 4 °C. However, the analytes are not quantitatively stored for 24 h of storage. After the storage of the analyte in the extraction needle, no new peak was obtained in the chromatogram Based on these results, extracted samples should be analyzed within 6 h.

Table 2

Storage recoveries of extracted amines in the extraction needle

Storage period (d)

Storage temperature (°C)

Storage recovery (%)

Ammonia

MA

DMA

TMA

3 h

25

96

94

104

105

6 h

25

97

96

94

109

24 h

25

49

79

81

79

24 h

4

49

82

67

75

Sample concentration: ammonia, MA, DMA 2 µg L−1; TMA 1 µg L−1

Sampling volume: 100 mL

Real Sample Analysis

Volatile amines generated from raw fish and raw meat were determined by the proposed method. The samples, a piece of raw codfish (86 g) and sliced pork (loin and belly; about 50 g each) on a glass petri dish, were individually placed in a gas sampling bag (Smart bag PA, 1 L volume). These sampling bags were then sealed with clips. The bags were then filled with clean ambient air and stored at 4 °C. Air samples (10 mL (fish sample) and 50 mL (meat sample))were collected from the sample headspace twice a day, and same volume of ambient air was introduced into the bag after the second sampling. Figure 6a shows the variation in the concentration of TMA in raw fish sample. The TMA concentration increases with date and reaches a maximum after a storage period of 9 days due to bacteria growth [27]. A typical chromatogram for the determination of TMA from the fish sample headspace is shown in Fig. 6b. During this period, other amines were not detected. Variation of the concentration of ammonia in meat samples are shown in Fig. 7a, a typical chromatogram for the determination of ammonia in pork loin sample is depicted in Fig. 7b. The ammonia concentration was lower than LOQ until storage period of 3 and 9 days for loin and belly samples, respectively, and increases with date due to spoilage of meat [28]. As shown in these results, the proposed method could be useful for evaluation of food spoilage.

Fig. 6

Determination of TMA in fish sample. a Variation of TMA concentration and b typical chromatogram for the determination of TMA (9 days storage)

Fig. 7

Determination of ammonia in meat samples. a Variations of ammonia concentration and b typical chromatogram for the determination of ammonia in pork loin (30 days storage)

Conclusion

The proposed analytical method using a needle-type extraction device and GC–BID offers simply and sensitive determination of low-molecular-weight amines in air samples. TMA is not affected by humidity, and is sensitively determined in real samples. However, the quantification of ammonia is affected by sample humidity. The proposed method could be further applied to the determination of volatile amines, such as those in food and drink samples. In addition, the developed method may be further developed for the sensitive determination of low-molecular-weight organic compounds such as alcohols and aldehydes.

Notes

Acknowledgements

This research was financially supported by the Shimadzu Science Foundation and JSPS KAKENHI (Grant Numbers: 15K1785, 18K05169).

Compliance with Ethical Standards

Conflict of interest

All authors declare that they have no conflict of interest.

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Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Department of Applied ChemistryUniversity of YamanashiKofuJapan
  2. 2.Shinwa Chemical Industries Ltd.KofuJapan
  3. 3.Department of Environmental and Life SciencesToyohashi University of TechnologyToyohashiJapan

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