Pre-column Derivatization-High Performance Liquid Chromatography for the Determination of Aliphatic Amines with Fluorescence Detection and Mass Spectrometry Identification

CHINESE JOURNAL OF ANALYTICAL CHEMISTRY Volume 35, Issue 6, June 2007 Online English edition of the Chinese language journal

Cite this article as: Chin J Anal Chem, 2007, 35(6), 779–785.

RESEARCH PAPER

Pre-column Derivatization-High Performance Liquid Chromatography for the Determination of Aliphatic Amines with Fluorescence Detection and Mass Spectrometry Identification Zhao Xian-En1,3, Li Yu-Lin1,3, You Jin-Mao1,2, Liu Yong-Jun1, Suo You-Rui1,* 1

Northwest Plateau Institute of Biology, Chinese Academy of Sciences, Xining 810001, China College of Chemistry Science, Qufu Normal University, Qufu 273165, China 3 Graduate School of the Chinese Academy of Sciences, Beijing 100039, China 2

Abstract: A simple and highly sensitive method based on the derivatization of aliphatic amines with 2-(2-phenyl-1H-phenanthro[9,10-d]imidazole-1-yl)-acetic acid (PPIA) followed by high-performance liquid chromatography (HPLC) with fluorescence detection and on-line post-column mass spectrometric identification has been developed. Optimum derivatization, giving the corresponding stable fluorescent derivatives, was obtained by the reaction of aliphatic amines with PPIA at 60 ℃ for 15 min in the presence of N-ethyl-N’-(3-dimethylamino propyl)carbodiimide hydrochloride (EDC) catalyst in acetonitrile solvent. The fluorescence detection wavelength of PPIA derivatives was 380 nm (260 nm of excitation wavelength). Separation of the derivatized amines with a good baseline resolution in conjunction with a gradient elution was carried out on a reversed phase Eclipse XDB-C8 column (4.6 mm × 150 mm, 5 μm), in this way, the contents of aliphatic amines in waste water of paper mill, telencephalon tissue of rat and acidophilus milk were determined. The identification of aliphatic amine derivatives was carried out by post-column online mass spectrometry with atmospheric pressure chemical ionization (APCI) source in positive ion mode. In virtue of the MS and MS/MS information of intermediates of derivatization reaction, the derivatization reaction mechanism was confirmed. The established method exhibits excellent reproducibility and recovery. Excellent linear responses were observed with coefficients >0.9996, and detection limits (at signal-to-noise of 3:1) were 3.1–18.2 fmol. Key Words: High performance liquid chromatography (HPLC/MS); Fluorescence detection; Pre-column derivatization; Aliphatic amines

1

Introduction

Aliphatic amines are widely distributed in nature and are paid much attention for their toxicity and reaction activity. Most amine compounds including aliphatic amines may occur as biodegradation products of organic matter such as proteins, amino acids, and other nitrogen-containing organic compounds. Volatile amines may react with nitrosating agents, leading to the formation of potentially carcinogenic N-nitrosamine compound[1,2]. Therefore, the determination of aliphatic amines at trace levels was highly significant for clinical science, detection and control of environment, food

testing and biology and so on. Most amine compounds show neither natural UV absorption nor fluorescence. At present, one of the most popular techniques used to improve detection limits is pre-column or post-column derivatization. A large number of derivatization reagents have been developed for the labeling of amine compounds, but at the same time, some shortcomings of these derivatization reagents in the determination of amines were also reported. For example, orthophthalaldehyde (OPA) method had poor reproducibility and stability of derivatives was dissatisfying[3]; 7-chloro-4nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) had poor stability and

Received 25 October 2006; accepted 2 February 2007 * Corresponding author. Email: [email protected] Copyright © 2007, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences. Published by Elsevier Limited. All rights reserved.

Zhao Xian-En et al. / Chinese Journal of Analytical Chemistry, 2007, 35(6): 779–785

underwent about 30%–50% decomposition in methanol-water solution within 25 min when exposed to daylight[4]; 6-Aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC)[5] was developed as a popular pre-column derivatization reagent for the determination of amines with satisfactory results. However, only 10% of the fluorescent intensity in aqueous solution compared with that in pure acetonitrile solution was observed for its derivatives. Thus, the detection limits for the early-eluted amine derivatives were usually higher; 9-Fluorenylmethyl chloroformate (FMOC)[6,7] and 1,2-benzo3,4-dihydrocarbazole-9-ethyl chloroformate[8] have been used for the derivatization of amines, amino acids, and peptides. For the effective derivatization, an excess reagent must be used, and derivatized solution must be extracted with pentane to remove excess of reagent because it interfered with the separation of derivatives, as a result, sometimes it resulted in some loss of their hydrophobic derivatives. Furthermore, these reagents were difficult to synthesize and easy to decompose. Based on the fact that fluorescence derivatization reagents with hydrazine group could label fatty acids sensitively with EDC as coupling reagent[9] and previous work of authors[10], 2-(2-phenyl-1H-phenanthro[9,10-d]imidazole-1-yl)-acetic acid was synthesized, which was more sensitive and more easily protonated in MS detection. Aliphatic amine derivatives were separated with a good baseline resolution in conjunction with a gradient elution and identified by post-column online mass spectrometry with APCI source in positive ion mode. The sensitivity, stability, simple derivatization reaction of PPIA with amines, and its applications for the determination of aliphatic amines from real samples were satisfactory.

2 2.1

pure acetonitrile was purchased from Merck (Germany). Water was purified on a Milli-Q system (Millipore, Bedford, MA, USA). All other reagents used were of analytical grade unless otherwise stated. DMF was redistilled prior to use. 2.2

Experimental methods

2.2.1

Preparation of standard solutions

Individual stock solution of the amines was prepared in acetonitrile. The standard amines for HPLC analysis at individual concentration of 1.0 × 10–4 M were prepared by diluting the corresponding stock solutions (0.01 M) of each amine with acetonitrile. The derivatizing reagent solution 2.0 × 10–3 M was prepared by dissolving 17.6 mg 2-(2-phenyl1H-phenanthro-[9,10-d]imidazole-1-yl)-acetic acid (PPIA) in 10 ml of spectroscopically pure acetonitrile, and to this solution was added 2 ml pyridine and diluted to 25 ml with acetonitrile. Standard solution of 0.15 M coupling reagent was prepared by dissolving 0.2876 g EDC.HCl in 10 ml of acetonitrile. When not in use, all standards were stored at 4℃. 2.2.2

Derivatization

To a solution consisting 40 μl of a standard amines mixture in a 2-ml vial, 40 μl EDC.HCl, and 120 μl derivatization reagent were successively added, respectively. The vial was then sealed and heated at 60℃ for 15 min in a thermostatic water-bath and then left to cool at room temperature. After the addition of 600 μl acetonitrile, 10 μl volume of the crude reaction solution was injected directly for chromatographic analysis. The derivatization procedure is shown in Fig.1.

Experimental Instruments and reagents

All the HPLC system devices were from the Agilent HP 1100 HPLC-MS series and consisted of a vacuum degasser (model G1322A), a quaternary pump (model G1311A), an autosampler (model G1329A), a thermostated column compartment (model G1316A), and a fluorescence detector (FLD) (model G1321A). Atmospheric pressure chemical ionization (APCI) source was in positive ion mode. Derivatives were separated on reversed-phase Eclipse XDB-C8 column (150 mm × 4.6 mm, 5 μm, Agilent). Fluorescence excitation and emission spectra were obtained at a 650-10 S fluorescence spectrophotometer (Hitachi). 2-(2-Phenyl-1H-phenanthro[9,10-d]imidazole-1-yl)acetic acid, 2-phenyl-1H-phenanthro[9,10-d] imidazole, 1,2-benzo-3,4dihydrocarbazole, and acridone were synthesized in authors’ laboratory. All aliphatic amine standards (C1–C12) and N-ethyl-N’-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) were purchased from Sigma Co. Spectroscopically

Fig.1 Derivatization scheme of PPIA with aliphatic amines

2.2.3

HPLC and MS conditions

Derivatives were separated on a reversed-phase Eclipse XDB-C8 column (150 mm × 4.6 mm, 5 μm, Agilent) by gradient elution. Eluent A was 30% acetonitrile consisting 30 mM formic acid (pH = 3.7) and B was 100% acetonitrile. The flow rate was constant at 1.0 ml min–1 and the column temperature was set at 30℃. The fluorescence excitation and emission wavelengths were set at λex = 260 nm and λem = 380 nm, respectively. Gradient conditions were: initial = 100% A at 35 min = 100% B (kept for 5 min, injection 10 μl). Before injection of the next sample, the column was equilibrated with mobile phase A for 10 min.

Zhao Xian-En et al. / Chinese Journal of Analytical Chemistry, 2007, 35(6): 779–785

Atmospheric pressure chemical ionization (APCI) was in positive-ion mode, and nebulizer pressure, dry gas temperature, dry gas flow rate, APCI Vap temperature, capillary voltage and corona current were 0.414 MPa, 350 ℃, 5.0 l min–1, 450 ℃, 3500 V, and 4000 nA (pos)[11], respectively. 2.2.4 Pretreatment of wastewater, telencephalon of male wistar rat, and acidophilus milk To a solution containing 50 ml of wastewater in 100-ml round-bottom flask, 2.0 ml hydrochloric acid (3.0 M) was added. The contents of the flask were vortexed for 2 min and filtered. The resultant solution was evaporated to dryness with a stream of nitrogen gas. The residue was re-dissolved with 50% acetonitrile solution consisting 0.2 M borate buffer (pH = 9.0) to a total volume of 10 ml and stored at 4 ℃ until HPLC analysis. Male wistar rats (about 300 g) were decapitated, the whole brain was removed, and washed using ice-cooled physiological salt solution. Telencephalon was separated onto a glass plate. To a hard glass tube, 1.2 ml 0.1 M ice-cooled HClO4 solution and the weighted telencephalon (about 2.0 g) were added, and slurried for 20 min. The slurried solution was centrifuged (18000 rpm for 30 min at 4 ℃). The supernatant solution was separated and 4 M NaOH was added to neutralize the excess of HClO4 to pH 6.5–7.0. The resultant solution was made up to a total volume of 1.5 ml with deionized water and stored at –80 ℃ until HPLC analysis. To a solution containing 2 ml of acidophilus milk in a 10-ml centrifugal tube, 2.0 ml deionized water, and 0.5 ml sodium hydroxide (4 M) were added. After 20 ml chloroform was added and sonicated for 10 min, the supernatant chloroform layer was extracted with an injector. This chloroform was adjusted to pH = 3.0 with 1 ml hydrochloric acid (4 M), and then evaporated to dryness with a stream of nitrogen gas. The residue was re-dissolved in 1.0 ml of a solution (ACN:H2O = 4:1, v:v), and stored at –10 ℃ until HPLC analysis. 2.3 2.3.1 zole

Synthesis of derivatization reagents (PPIA) Synthesis of 2-phenyl-1H-phenanthro[9,10-d]imida-

The synthesis of 2-phenyl-1H-phenanthro[9,10-d]imidazole was carried out according to the method previously reported[12]: 9,10-Phenanthraquinone (16 g), benzaldehyde (10 ml), and ammonium acetate (120 g) were fully mixed in 500-ml of round-bottom flask. After the addition of 300 ml glacial acetic acid, the contents were rapidly heated to 80–90℃with vigorous stirring for 3 hours. After cooling, the contents were moved to a beaker with 300 ml water, and pH of solution was adjusted to 7–8 with ammonia water. The

precipitated solid was recovered by filtration, washed with water, and dried at room temperature for 48 hours. The crude product was recrystallized twice from acetonitrile/DMF mixed solvent (acetonitrile/DMF 5:1, v/v) to afford a slight yellow crystal, yield 90%. 2.3.2 Synthesis of 2-(2-phenyl-1H-phenanthro[9,10-d] imidazole-1-yl)acetic acid 2-(2-Phenyl-1H-phenanthro[9,10-d]imidazole-1-yl)acetic acid was synthesized as follows:. 2-Phenyl-1H-phenanthro [9,10-d]imidazole (28 g), KOH (30 g) and dimethylsulfoxide (250 ml) were mixed in a 500-ml round-bottom flask and rapidly heated to 120–125 ℃ for 10 min with vigorous stirring. A solution of 50 ml ethyl bromoacetate in 30 ml dimethylsulfoxide was added dropwise within 2.5 hours with stirring. After cooling, 500 ml water and 20 g KOH were added and continuously heated at 100 ℃ for 20 min. After the solution was cooled to room temperature, the mixture was adjusted to pH=8.0, filtered removing the impurity, and the resultant solution was neutralized to pH 3.0 with 4 M HCl solution. The precipitated solid was recovered by filtration. The crude product was dried at room temperature for 48 hours and recrystallized twice from acetonitrile to afford a white crystal, yield 84%. m.p. >300 ℃. Found: C 78.32; H 4.58; N 7.94. Calculated: C 78.39; H 4.57; N 7.95. IR (KBr): 3110.57(–OH, dimer), 1711.82 (C=O), 1657.09, 1612.53 (ph–C=N–), 1529.62, 1474.14, 1460.11, 1424.59 (ph), 1399.26 (C-H), 1229.85, 1111.75, 773.37, 750.7, 720.84, 703.84. m/z [M + H]+ 353.1.

3

Results and discussion

3.1 Stability and spectrum properties of PPIA PPIA was synthesized in dimethylsulfoxide solvent; after washing with water and recrystallization in acetonitrile, it was stable in water or in common organic solvents. When an anhydrous solution of PPIA in acetonitrile was stored under room temperature for two weeks, the derivatization yields for amines were not obviously different. The fluorescence excitation and emission wavelengths were set at λex = 260 nm and λem = 380 nm, respectively. According to Lambert-Beer’s Law (A = εbc, A: absorbance, ε: molar absorptivity, b: colorimetric tube, commonly 1 cm，c: concentration of solution, M ), the molar absorptivity of PPIA in acetonitrile at maximal ultraviolet wavelength (251 nm) was 6.3 × 105 l mol–1 cm–1. The molar absorptivities and molecular structure of PPIA and four fluorescence derivatization reagents (AAA[10], BCEOC[8], CEOC[13], FMOC[13]) are shown in Table 1. The molar absorptivity (ε) at maximal ultraviolet absorption wavelength was close to the detection sensitivity[14], and in Table 1 the molecular conjugated degree and the molar absorptivity (ε) of

Zhao Xian-En et al. / Chinese Journal of Analytical Chemistry, 2007, 35(6): 779–785

PPIA were all the maximal, so the molecular structure of fluorescence derivatization reagent was the structure foundation and is the reason for their high detection sensitivity. This may provide basement and guidance for the synthesis of sensitive fluorescence derivatization reagent. Table 1 Comparison of molar absorptivity for PPIA with the other four fluorescence derivatization reagents Derivatization reagent

Molar absorptivity ε (l mol–1 cm–1)

Molecular structure

N

PPIA

N CH 2 COOH

6.3 × 105

CH2COOH N

3.2 Optimal derivatization The derivatization yields for PPIA and amines were obviously different at various derivatization time periods, temperatures, and concentrations of PPIA and EDC. The optimal derivatization conditions were as follows: most high derivatization yield was found at 60 ℃, above it there were some by-products and the derivatization yield decreased. Maximum and constant peak intensities could be attained with derivatization time at 15 min. With a further long derivatization time, the detector responses did not significantly increase. The constant fluorescence intensity was achieved with the addition of 5-fold molar reagent excess to total molar amines and it had no significant effect on yields by increasing the excess of reagent beyond this level. For the coupling reagent EDC, 40 μl of EDC (0.15 M, 25-fold molar excess to PPIA) was enough for complete derivatization.

2.9 × 105

AAA

3.3 Derivatization mechanism

O

O CH2CH2O

BCEOC

Cl 4

2.4 × 10

N

O

CEOC

CH2CH2O N

Cl

2.3 × 104

O CH2CH2O

Cl

FMOC

1.7 × 104

PPIA: (2-(2-phenyl-1H-phenanthro-[9,10-d]imidazole-1-yl)-acetic acid); AAA: (2-(9-acridone)-acetic acid); BCEOC: (1,2-benzo-3,4-dihydrocarbazole-9-ethyl chloroformate); CEOC: (2-(9-carbazole)-ethyl chloroformate); FMOC: (2-(9fluorenyl)-ethyl chloroformate).

Based on the derivatization mechanism of fluorescence labeling reagent containing hydrazine with fatty acids[9], the derivatization mechanism of PPIA and amines was guessed theoretically as follows: first, PPIA reacted with amines to create active intermediate, and second, amines reacted with the active intermediate to give the stable fluorescent derivatives. Peak A and B in the chromatogram (Fig.5) of standard amine derivatives were identified by online post column mass spectrometry, the MS and MS/MS information about specific fragment ions about peak A and B were analyzed, and peak A and B were confirmed as the active intermediates. Mass spectrum of intermediates A and B was shown in Fig.2, MS cleavage mode of intermediates A and B was shown in Fig.3, and derivatization mechanism of PPIA with amines was shown in Fig.4.

Fig.2 Mass spectrum of intermediates A and B a and b is molecular ion MS and MS/MS belonging to intermediate A; c and d is molecular ion MS and MS/MS belonging to intermediate B

3.4 Contrast of MS Ion current intensity

Under the same experimental conditions, the MS ion current intensity of 2-phenyl-1H-phenanthro-[9,10-d]imidazole (PPI),

Zhao Xian-En et al. / Chinese Journal of Analytical Chemistry, 2007, 35(6): 779–785

1,2-benzo-3,4-dihydrocarbazole (BDC), and acridone was contrasted by injecting the same amounts of them into mass spectrometer. As a result, IPPI : IAcridone : IBDC = 12:5:1 (IPPI, IBDC and IAcridone were the MS ion current intensity of PPI, BDC and

Acridone, respectively). Thus, PPI had highest MS protonation efficiency, and this might contribute to the fact that PPI molecule contains two nitrogen atoms with strong electronegative effect.

Fig.3 Scheme of MS cleavage mode of intermediates A and B

Fig.4 Derivatization mechanism scheme of PPIA with amines

Fig.5 Chromatogram (a) and MS spectrum of total ion current (b) for standard amines derivatized with PPIA Chromatographic conditions as described in experimental section. C1 (methylamine); C2 (ethylamine); C3 (propylamine); C4 (butylamine); C5 (pentylamine); C6 (hexylamine); C7 (heptylamine); C8 (octylamine); C9 (nonylamine); C10 (decylamine); C11 (undecylamine); C12 (dodecylamine); A and B (intermediates ); C (2-phenyl-1H-phenanthro-[9,10-d]imidazole)

Zhao Xian-En et al. / Chinese Journal of Analytical Chemistry, 2007, 35(6): 779–785

3.5

Chromatographic separation and MS analysis

A good baseline resolution was achieved by adjusting the gradient elution conditions and the pH of eluent A. Because of the two alkalescence nitrogen atoms in the molecular core structure of PPIA, by controlling the pH of eluent A at 3.7 with formic acid, the simultaneous separation of 12 amine derivatives was achieved with the shortest retention times and

the sharpest peaks. Additionally, an acidic mobile phase would be useful to the ionization of PPIA-amines Chromatogram and MS spectrum of total ion current for standard amines derivatized with PPIA were shown in Fig.5. PPIA-amine derivatives were identified by online post column mass spectrometry with APCI source in positive ion mode. The MS data are shown in Table 2, and typical MS chromatogram of representative decylamine derivative are shown in Fig.6.

Fig.6 Typical MS chromatogram of representative decylamine derivative (a, molecular ion MS; b, MS/MS) Table 2 Linear regression equations, correlation coefficients, detection limits, MS of aliphatic amine derivatives, and repeatability for peak area and retention time (n = 6) Amine C1 C2 C3 C4 C5 C6 C7 C8 C9 C10 C11 C12

3.6

Y = AX + B Correlation coefficient X: Injected amount (pmol), Y: Peak area r Y = 20.03X – 3.628 Y = 25.59X + 5.556 Y = 56.41X + 7.253 Y = 52.42X + 14.20 Y = 58.33X + 16.32 Y = 65.67X + 16.25 Y = 65.24X + 15.51 Y = 78.75X + 20.55 Y = 109.9X + 27.16 Y = 107.5X + 22.94 Y = 101.8X + 22.75 Y = 141.3X + 35.28

0.9996 0.9985 0.9999 0.9996 0.9996 0.9998 0.9997 0.9997 0.9997 0.9998 0.9997 0.9997

Stability of derivatives

A solution at room temperature containing 50 pmol standard PPIA-amine derivatives was analyzed by HPLC at 0, 1, 2, 4, 8, 16, 24 and 72 hours after derivatization. The standard deviations of all the amine derivatives calculated by peak area relative to the value of 0 hour were all less than 2.8%, and thus the stability of PPIA-amine derivatives was satisfactory for chromatographic analysis. 3.7 Repeatability, linear regression equations and detection limits

Detection limits (fmol)

MS [M+1]+

Retention time RSD (%)

Peak area RSD (%)

18.21 16.48 7.55 6.84 7.61 5.14 6.14 4.82 3.80 4.51 4.18 3.08

366.1 380.1 394.1 408.2 422.2 436.2 450.3 464.3 478.3 492.3 506.3 520.3

0.094 0.090 0.091 0.064 0.072 0.063 0.058 0.062 0.050 0.049 0.050 0.043

2.10 1.89 2.32 2.65 1.83 1.16 0.79 0.73 0.63 0.61 0.62 0.61

2.7%. Based on the optimum derivatization conditions, the calibration graphs were established with the peak area (Y) versus amines quantities (X, pmol, corresponding injected amount from 200.0 pmol to 48.83 fmol). Linear regression equations, correlation coefficients, and detection limits for all amine derivatives were shown in Table 2. All amine derivatives were found to give excellent linear responses over this range with correlation coefficients of 0.9985–0.9999. The calculated detection limits of each amine (at a signal-to-noise ratio of 3:1) were from 3.1–18.2 fmol.

4 Under the same optimum chromatographic conditions, a standard solution consisting 50 pmol C1–C12 amine derivatives was prepared for the examination of the method repeatability. The relative standard deviations (RSDs) of the peak areas and retention times are shown in Table 2. RSDs of retention time were less than 0.10%, and RSDs of peak area were less than

4.1

Analysis of samples Recovery

A known amount of aliphatic amines (10 μl, 1.0 × 10–4 M) was added into 50 ml wastewater of paper mill in which the contents of amines had been determined, while the extraction

Zhao Xian-En et al. / Chinese Journal of Analytical Chemistry, 2007, 35(6): 779–785

and derivatization were the same as described above. The analyses were carried out thrice, and the recoveries for 12 aliphatic amines were 86.6%–105.1% (Table 3). 4.2

Determination of samples

were according to the optimum conditions above, and the derivatives of amines in real samples were identified by online post column mass spectrometry. Chromatogram of aliphatic amines from waste water of paper mill (a), telencephalon tissue of Wistar rat (b) and acidophilus milk (c) were shown in Fig.7, and the contents of amines from them are shown in Table 3.

Derivatization and chromatographic separation conditions

Fig.7 Chromatogram of free aliphatic amines from waste water of paper mill (a), telencephalon tissue of Wistar rat (b) and acidophilus milk (c) Table 3 Contents of aliphatic amines from real samples and recoveries (n = 3) Amine

Waste water of Paper mill (μg l–1)

CH3NH2 CH3CH2NH2 CH3(CH2)2NH2 CH3(CH2)3NH2 CH3(CH2)4NH2 CH3(CH2)5NH2 CH3(CH2)6NH2 CH3(CH2)7NH2 CH3(CH2)8NH2 CH3(CH2)9NH2 CH3(CH2)10NH2 CH3(CH2)11NH2

Telencephalon tissueof Wistar rat (μg g–1)

Acidophilus milk (μg l–1)

Recoveries (%) of waste water

8.62 9.87 7.94 6.36 1.21

6.13 2.51 0.61 0 0

2.32 1.08 0.84 0 0

86.6 92.6 97.8 105.1 102.6

7.61 1.21 2.03 1.81 1.52 2.19 2.94

0 0.58 0.043 0.013 0 0 0

0 0.46 0.16 0.14 0 0 0

93.7 99.9 104.4 89.2 91.9 101.3 104.4

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