Abstract: A new fluorescent derivative reagent 2O(9Oacridone) O acetic acid (AAA) was used for pre-column derivatization and high performance liquid chromatography ( HPLC ) separation and linear mass spectrometry for fatty amines by fluorescence detection. Derivative fluorescence excitation and The emission wavelength is λex = 404 nm, λem = 440 nm. At 30 °C, NO ethyl ON'O[(3Odimethylamino)propyl]carbodiimide hydrochloride (EDC) is used as a catalyst in acetonitrile solvent. A stable fluorescent product was obtained after 20 min of derivatization. On the Hypersil BDS C18 (4.6 mm × 100 mm, 5 μm) column, 12 aliphatic amine derivatives were optimized by gradient elution. Atmospheric pressure chemical ionization Source (APCI Source) positive ion mode for on-line post-column mass spectrometry, to achieve a rapid and accurate determination of various fatty amine derivatives. The method has good reproducibility, the linear regression coefficient of most fatty amines is greater than 0. 999 6 , detection limit is 12. 09 ~ 25. 52fmol.
Key words: high performance liquid chromatography-mass spectrometry; fluorescence detection; pre-column derivatization; amine compounds present in the fatty amine environment have become a class of compounds that have received much attention due to their toxicity and reactivity. It is of great significance to environmental chemistry, biology, toxicology and clinical chemistry. Fatty amines are usually compounds without UV absorption or non-luminescence properties. Chemical derivatization is an effective way to improve the detection sensitivity of such compounds. The compounds have more fluorescent reagents [1 - 4 ], but most of the reagents have defects in the application. It is still necessary to continue to develop reagents with high sensitivity and simplicity. The highly sensitive fatty acids are derived from the thiol-based fluorescent reagents under the action of EDC. Inspired [ 5 - 7 ], and acridone [ 8 ] has strong fluorescence intensity. The authors synthesized 2O (9O acridone) O acetic acid (AAA) pre-column derivatization reagent, and used EDC in acetonitrile solvent at 30 °C. Complete derivatization of fatty amines can be achieved within 20 min of catalyst. The obtained product is stable and the yield is high. The derivatized solution can be directly injected without treatment. The acetonitrile/water is used.
1 Experimental part
1. 1 Instruments and reagents Agilent 1100 high performance liquid chromatography-mass spectrometer (Agilent) equipped with quaternary gradient pump, online vacuum degasser, fluorescence detector, atmospheric pressure chemical ionization source, Hypersil BDS C18 column (4. 6 mm × 100 mm, 5 μm, Dalian Elite Co., Ltd.. 2O (9O acridone) O acetic acid (homemade), 12 fatty amine standard samples (Sigma), EDC (Sigma), spectral grade acetonitrile (Merck) ), formic acid, N, NO dimethylformamide (DMF) are analytically pure, pure water is prepared by MilliOQ ultrapure water system.
1. 2 Preparation of the standard solution Accurately take a quantitative standard of the fatty amine, using a spectrum of pure acetonitrile to form a solution of 1.0 × 10 - 2 mol / L, corresponding to a low concentration of fatty amine standard solution (1. 0 × 10 - 4 mol / 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。. 1摩尔的溶液。 0. 191 7 mg of NO ethyl ON ′O [(3O dimethylamino) propyl] carbodiimide hydrochloride (EDC) dissolved in 10mL of spectrally pure acetonitrile to form a solution of 0.1 mol / L .
1. 3 Derivatization of fatty amines In a 2 mL ampule, 45 μL of EDC, 40 μL of a fatty amine standard sample, and 80 μL of a derivatized reagent solution were added, sealed, and reacted in a 30 ° C water bath for 20 min. The derivatization reaction is shown in Figure 1.
1. 4 Chromatography and mass spectrometry conditions Chromatographic conditions: mobile phase A: 10% aqueous acetonitrile; B: 100% acetonitrile (containing 2.5% DMF). B gradient from 0% to 100% in 35 min, flow rate 1 0 mL / min, injection volume 10 μL, column temperature 30 ° C. Fluorescence excitation and emission wavelengths are: λex = 404 nm, λem = 440 nm.
Mass spectrometry conditions: atmospheric pressure chemical ionization source, positive ion mode, spray pressure 60p. s. i (1p. s. i = 6894. 76 Pa), dry gas flow rate 5L / min, dry gas temperature 350 °C, gasification temperature 400 °C, capillary voltage 3 500 V, corona current 4 000 nA ( Pos) [ 9, 10 ] .
2 Results and discussion
2. 1 Optimization of the derivatization conditions 2O (9O acridone) O acetic acid derivatization reaction of fatty amines with the difference of the time and temperature of the derivatization reaction and the different yields of the derivatization reagent and EDC dosage, with ethylamine For example, hexylamine, decylamine and dodecylamine were optimized for the above four factors. The results show that, under certain other factors, there is a high yield of zui at 30 °C, and a side reaction with increasing temperature. The productivity decreased, and the signal intensity of the derivative product was stable after 20 min of derivatization reaction. The authors chose to derivatize the reaction at 30 °C for 20 min. After the derivatization reagent reached 6 times the total amount of fatty amine, the signal intensity of the product was basically stable, and the yield of EDC was 45 μL. Zui high, as shown in Figure 2.
2. 2 Chromatographic Separation and Mass Spectrometric Identification of Standards On the HypersilBDS C18 (4.6 mm × 100 mm, 5 μm) column, acetonitrile/water was used as the mobile phase, and 12 species were realized in 35 min by gradient elution (C1 - C12) Complete baseline separation of linear aliphatic amine derivatives. The results are shown in Figure 3. The on-line post-column mass spectrometry was performed using an atmospheric pressure chemical ionization source (APC I Source) positive ion mode. The mass spectrometry data of each component are shown in Table 1.
2. 3 linear regression equation and detection limit injection volume in the range of 200. 0 pmol ~ 48. 83 fmol, according to the peak area and the actual injection volume linear regression, the regression equation of each fatty amine derivative obtained. Correlation coefficient and The detection limit is shown in Table 1. The linear correlation coefficient of each fatty amine derivative is 0. 999 6 or more, and the detection limit is between 12. 09 and 25. 52 fmol (by S/N). = 3:1 calculation).
2. 4 Reproducibility experiments Under the same elution conditions, 50 pmol of fatty amine derivatives were analyzed in parallel for six times. The retention time and peak area reproducibility are shown in Table 2. The relative standard deviation (RSD) of retention time is less than 0. 9%。 The relative standard deviation of the peak area RSD is less than 2. 9 %.
3 氯 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 20 s to convert it into the corresponding organic salt of the fatty amine, the solvent was evaporated to dryness under reduced pressure, and dissolved in 3.0 mL of 80% acetonitrile/water solution for storage. The soil extract was chromatographed according to the above experimental conditions, and the content of fatty amine was The determination results are shown in Table 3. After adding a certain amount of fatty amine standard in the soil sample, the extract is extracted according to the above extraction method, and the recovery is shown in Table 3. Conclusion: Using 2O (9O acridone) O acetic acid (AAA) As a pre-column derivatization reagent, the derivatization and chromatographic conditions were optimized, and a sensitive and rapid method for the determination of fatty amines was established. The derivatization yield of reagents and fatty amines was high and the product was stable. The actual sample measurement results showed that the established The method has a wide linear range and good reproducibility.
References (omitted)
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Key words: high performance liquid chromatography-mass spectrometry; fluorescence detection; pre-column derivatization; amine compounds present in the fatty amine environment have become a class of compounds that have received much attention due to their toxicity and reactivity. It is of great significance to environmental chemistry, biology, toxicology and clinical chemistry. Fatty amines are usually compounds without UV absorption or non-luminescence properties. Chemical derivatization is an effective way to improve the detection sensitivity of such compounds. The compounds have more fluorescent reagents [1 - 4 ], but most of the reagents have defects in the application. It is still necessary to continue to develop reagents with high sensitivity and simplicity. The highly sensitive fatty acids are derived from the thiol-based fluorescent reagents under the action of EDC. Inspired [ 5 - 7 ], and acridone [ 8 ] has strong fluorescence intensity. The authors synthesized 2O (9O acridone) O acetic acid (AAA) pre-column derivatization reagent, and used EDC in acetonitrile solvent at 30 °C. Complete derivatization of fatty amines can be achieved within 20 min of catalyst. The obtained product is stable and the yield is high. The derivatized solution can be directly injected without treatment. The acetonitrile/water is used.
1 Experimental part
1. 1 Instruments and reagents Agilent 1100 high performance liquid chromatography-mass spectrometer (Agilent) equipped with quaternary gradient pump, online vacuum degasser, fluorescence detector, atmospheric pressure chemical ionization source, Hypersil BDS C18 column (4. 6 mm × 100 mm, 5 μm, Dalian Elite Co., Ltd.. 2O (9O acridone) O acetic acid (homemade), 12 fatty amine standard samples (Sigma), EDC (Sigma), spectral grade acetonitrile (Merck) ), formic acid, N, NO dimethylformamide (DMF) are analytically pure, pure water is prepared by MilliOQ ultrapure water system.
1. 2 Preparation of the standard solution Accurately take a quantitative standard of the fatty amine, using a spectrum of pure acetonitrile to form a solution of 1.0 × 10 - 2 mol / L, corresponding to a low concentration of fatty amine standard solution (1. 0 × 10 - 4 mol / 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。. 1摩尔的溶液。 0. 191 7 mg of NO ethyl ON ′O [(3O dimethylamino) propyl] carbodiimide hydrochloride (EDC) dissolved in 10mL of spectrally pure acetonitrile to form a solution of 0.1 mol / L .
1. 3 Derivatization of fatty amines In a 2 mL ampule, 45 μL of EDC, 40 μL of a fatty amine standard sample, and 80 μL of a derivatized reagent solution were added, sealed, and reacted in a 30 ° C water bath for 20 min. The derivatization reaction is shown in Figure 1.
1. 4 Chromatography and mass spectrometry conditions Chromatographic conditions: mobile phase A: 10% aqueous acetonitrile; B: 100% acetonitrile (containing 2.5% DMF). B gradient from 0% to 100% in 35 min, flow rate 1 0 mL / min, injection volume 10 μL, column temperature 30 ° C. Fluorescence excitation and emission wavelengths are: λex = 404 nm, λem = 440 nm.
Mass spectrometry conditions: atmospheric pressure chemical ionization source, positive ion mode, spray pressure 60p. s. i (1p. s. i = 6894. 76 Pa), dry gas flow rate 5L / min, dry gas temperature 350 °C, gasification temperature 400 °C, capillary voltage 3 500 V, corona current 4 000 nA ( Pos) [ 9, 10 ] .
2 Results and discussion
2. 1 Optimization of the derivatization conditions 2O (9O acridone) O acetic acid derivatization reaction of fatty amines with the difference of the time and temperature of the derivatization reaction and the different yields of the derivatization reagent and EDC dosage, with ethylamine For example, hexylamine, decylamine and dodecylamine were optimized for the above four factors. The results show that, under certain other factors, there is a high yield of zui at 30 °C, and a side reaction with increasing temperature. The productivity decreased, and the signal intensity of the derivative product was stable after 20 min of derivatization reaction. The authors chose to derivatize the reaction at 30 °C for 20 min. After the derivatization reagent reached 6 times the total amount of fatty amine, the signal intensity of the product was basically stable, and the yield of EDC was 45 μL. Zui high, as shown in Figure 2.
2. 2 Chromatographic Separation and Mass Spectrometric Identification of Standards On the HypersilBDS C18 (4.6 mm × 100 mm, 5 μm) column, acetonitrile/water was used as the mobile phase, and 12 species were realized in 35 min by gradient elution (C1 - C12) Complete baseline separation of linear aliphatic amine derivatives. The results are shown in Figure 3. The on-line post-column mass spectrometry was performed using an atmospheric pressure chemical ionization source (APC I Source) positive ion mode. The mass spectrometry data of each component are shown in Table 1.
2. 3 linear regression equation and detection limit injection volume in the range of 200. 0 pmol ~ 48. 83 fmol, according to the peak area and the actual injection volume linear regression, the regression equation of each fatty amine derivative obtained. Correlation coefficient and The detection limit is shown in Table 1. The linear correlation coefficient of each fatty amine derivative is 0. 999 6 or more, and the detection limit is between 12. 09 and 25. 52 fmol (by S/N). = 3:1 calculation).
2. 4 Reproducibility experiments Under the same elution conditions, 50 pmol of fatty amine derivatives were analyzed in parallel for six times. The retention time and peak area reproducibility are shown in Table 2. The relative standard deviation (RSD) of retention time is less than 0. 9%。 The relative standard deviation of the peak area RSD is less than 2. 9 %.
3 氯 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 20 s to convert it into the corresponding organic salt of the fatty amine, the solvent was evaporated to dryness under reduced pressure, and dissolved in 3.0 mL of 80% acetonitrile/water solution for storage. The soil extract was chromatographed according to the above experimental conditions, and the content of fatty amine was The determination results are shown in Table 3. After adding a certain amount of fatty amine standard in the soil sample, the extract is extracted according to the above extraction method, and the recovery is shown in Table 3. Conclusion: Using 2O (9O acridone) O acetic acid (AAA) As a pre-column derivatization reagent, the derivatization and chromatographic conditions were optimized, and a sensitive and rapid method for the determination of fatty amines was established. The derivatization yield of reagents and fatty amines was high and the product was stable. The actual sample measurement results showed that the established The method has a wide linear range and good reproducibility.
References (omitted)
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