Binary Fluorous Alkylation of Biogenic Primary Amines with

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Binary Fluorous Alkylation of Biogenic Primary Amines with Perfluorinated Aldehyde Followed by Fluorous Liquid Chromatography−Tandem Mass Spectrometry Analysis Tadashi Hayama,† Yohei Sakaguchi,† Hideyuki Yoshida,† Miki Itoyama,† Kenichiro Todoroki,‡ Masatoshi Yamaguchi,† and Hitoshi Nohta*,† †

Faculty of Pharmaceutical Sciences, Fukuoka University, 8-19-1 Nanakuma, Johnan, Fukuoka 814-0180, Japan Laboratory of Analytical and Bio-Analytical Chemistry, Graduate School of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Suruga, Shizuoka 422-8526, Japan

‡

S Supporting Information *

ABSTRACT: We have developed a novel method for the determination of biogenic amines (dopamine, norepinephrine, 3-methoxytyramine, normetanephrine, serotonin, tyramine, tryptamine, 5-methoxytryptamine, and histamine) utilizing liquid chromatography with electrospray ionization tandem mass spectrometry (LC−ESI-MS/MS) combined with a separation-oriented derivatization technique. Using this approach, primary amino groups in the target amines were selectively dialkylated with a perfluorinated aldehyde reagent (2H,2H,3H,3H-perfluoroundecan-1-al) through reductive amination. The derivatives were directly injected onto an LC column containing perfluoroalkyl-modified stationary phase and were separated via gradient elution using a water/methanol/ trifluoroacetic acid mixture and trifluoroethanol with formic acid as mobile phases. Matrix-induced signal suppression effects were eliminated because the binary fluorous-labeled amines were strongly retained on the fluorous-phase LC column, whereas the nonfluorous derivatives, including matrix components and monofluorous-labeled compounds such as the derivatization reagent, were poorly retained under the separation conditions. The linear dynamic ranges of the target amines were established over a concentration range of 0.01−1 nM (r > 0.9978), and the limits of detection were found to be 7.8−26 amol on column. The feasibility of this method was further evaluated by applying it to human plasma samples.

B

amino acid detection by ESI-MS, utilizing N-alkylnicotinic acid N-hydroxysuccinimide ester as a derivatization reagent.5 Shimbo et al. reported a sensitive LC−MS/MS method for the detection of amino acids following derivatization with a permanently charged reagent, p-N,N,N-trimethylammonioanilyl N′-hydroxysuccinimidyl carbamate iodide.8 These reagents are designed to include two isobaric forms, one for analytes and a second for standards. This type of differential isotope labeling technique has been used successfully for relative and absolute quantification of analytes. In addition, reductive amination chemistry has been utilized as a derivatization scheme for the LC−MS/MS analysis of amines.6,7 This reaction is simple, specific, and quantitative for analytes containing both an amine and an aldehyde. Guo et al. reported the derivatization of amine-containing metabolites using reductive amination with formaldehyde and succeeded in the analysis of differential isotope-labeled dimethylated amines with formaldehyde by reversed-phase or hydrophilic interaction LC coupled with Fourier transform ion cyclotron resonance MS.6 Ji et al.

iogenic amines, such as catecholamines, indoleamines, and histamine, play essential biological roles and are of great clinical importance in the diagnosis of some metabolic diseases and neoplasias.1−4 Simultaneous determination of these molecules in biological fluids provides critical information related to study of human diseases. However, because of their low basal levels in biological samples, a sensitive and selective determination method is required. Recently, the combination of chemical derivatization techniques with liquid chromatography−mass spectrometry (LC−MS) analysis has become an attractive approach for determining trace amounts of biogenic compounds.5−14 Permanently charged, proton affinity, and hydrophobic species have long been used as conventional derivatization reagents to enhance sensitivity in LC−MS analysis. One of the most frequently used derivatization reagents for amines is iTRAQ (isobaric tags for relative and absolute quantitation), which is often applied to amino acid, peptide, and protein analysis.9,10 Permanently charged quaternary ammonium containing derivatization reagents have been also very amenable for LC− MS analysis utilizing an electrospray ionization (ESI) source. Yang et al. reported a method combining derivatization with LC and tandem mass spectrometry (MS/MS) to enhance © 2012 American Chemical Society

Received: July 16, 2012 Accepted: September 5, 2012 Published: September 5, 2012 8407

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Figure 1. Derivatization reaction of primary amines via reductive amination with PFUA and 2-PB.

developed an LC−MS/MS method for their determination that eliminates matrix-induced effects. We previously reported an LC-fluorescence detection method for some natively fluorescent biogenic amines following derivatization with a fluorous isocyanate reagent, 2-(perfluorooctyl)ethyl isocyanate.32 This method was successfully applied to the simultaneous determination of biogenic amines in human urine without interference from contaminants in the biological matrix. To achieve a more sensitive, selective, and accurate analysis of biogenic amines in this study, we employed a fluorous aldehyde derivatization reagent, 2H,2H,3H,3H-perfluoroundecan-1-al (PFUA), via reductive amination followed by LC−MS/MS analysis. The primary amino groups of the target amines were easily dialkylated with PFUA in the presence of the nontoxic reductant 2-picoline borane, 2-PB (Figure 1). The resulting binary fluorous-labeled derivatives were detected with high sensitivity by ESI-MS/MS following selective separation based on their extreme fluorophilicity using an LC column packed with fluorous stationary phase. After analytical conditions for both the derivatization and LC−MS/MS analysis were optimized using standards of the target amines, the method was applied to the analysis of human plasma samples.

employed a diethylation method with acetaldehyde for the sensitive UPLC−MS/MS determination of monoamine neurotransmitters such as norepinephrine, dopamine, serotonin, and normetanephrine.7 Their reported limits of detection (LOD) were amazingly low, as the diethylated monoamines afforded 20−100 times increased detection sensitivity in MS analysis compared with their native forms. The methods previously described focus on highly sensitive detection-oriented derivatization for MS analysis. However, one common problem encountered in the LC−MS analysis of biological analytes is that complex matrixes often prevent accurate quantification of molecules of interest through matrixinduced ion enhancement/suppression effects arising from components endogenous to the matrix.15−19 These effects, thought to be caused by ionization competition between the analytes and coeluting components, are often observed in the analysis of complex matrix samples, especially in ESI-based MS. Various approaches have been utilized to overcome these matrix-induced effects,20−26 including the use of stable isotopelabeled internal standards, although such materials may not be available to all laboratories because their commercial availability is limited and because they may exhibit unexpected behavior during pretreatment or LC−MS analysis.20−22 Several effective approaches to sample preparation to remove matrix components have also been developed, almost all of which are based on solid-phase extraction techniques;23−26 but unfortunately, most are tedious and time-consuming. Alternatively, we proposed a specific separation-oriented derivatization method for eliminating matrix effects in LC−MS analysis.11 Perfluoroalkyl-containing compounds are highly fluorous, meaning that they have a remarkable affinity for one another and effectively exclude nonfluorous species.11,27−32 Therefore, analytes derivatized with fluorous compounds can be retained specifically on an LC column packed with perfluorinated stationary phase and clearly separated from early eluting nonfluorous components. This method relieves the necessity for further sample pretreatment or the use of stable isotope-labeled standards to correct for matrix effects. In the present study, we applied a fluorous derivatization method to biogenic primary amines (dopamine, norepinephrine, 3-methoxytyramine, normetanephrine, serotonin, 5-methoxytryptamine, tryptamine, tyramine, and histamine) and

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EXPERIMENTAL SECTION Reagents and Materials. Dopamine (DA) hydrochloride, norepinephrine (NE) bitartrate, serotonin (5-HT) hydrochloride, tyramine (Tyr) hydrochloride, tryptamine (Trp), 5methoxytryptamine (5-MT), and histamine (His) were purchased from Wako Pure Chemicals (Osaka, Japan). 3Methoxytyramine (3-MT) hydrochloride, normetanephrine (NM) hydrochloride, and 2,2,2-trifluoroethanol (TFE) were from Sigma-Aldrich (St. Louis, MO, U.S.A.). PFUA and 2-PB were obtained from Fluorous Technologies (Pittsburgh, PA, U.S.A.) and Tokyo Chemical Industry (Tokyo, Japan), respectively. Trifluoroacetic acid (TFA) was purchased from Kanto Chemical (Tokyo, Japan). Deionized water was purified using a Millipore EQG system (Billerica, MA, U.S.A.) and was used to prepare all solutions. All other organic solvents and acids from Wako Pure Chemical were of LC-grade and used as received. Derivatization Procedure. To 40 μL of sample solution placed in a 1.5 mL screw cap tube, 40 μL of isopropyl alcohol 8408

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was added, and the mixture was well-vortexed. After centrifugation at 10 000g for 10 min, a 70 μL aliquot of the sample was transferred to another tube, and 20 μL of 2.0 M PFUA in isopropyl alcohol and 10 μL of 1.5 M 2-PB in isopropyl alcohol were added. The mixture was incubated at room temperature for 15 min. Then 20 μL of 2.5 M diphenylamine in isopropyl alcohol was added in order to consume the excess reagents. The resulting solution was filtered using a PTFE filter (DISMIC-13HP, 0.2 mm; Advantec Toyo Kaisha, Tokyo, Japan) and placed in the autosampler of the LC−MS/MS system. Analysis of Human Plasma Sample. Plasma samples were obtained from whole blood donated by healthy volunteers in our laboratory. A centrifugal blood collection tube (Eiken Chemical, Tokyo, Japan) was used for the collection of the samples. After collection, the blood was immediately centrifuged at 2000g for 5 min at room temperature. The supernatant plasma was transferred to a screw-capped polypropylene tube and used without further processing. The sample was derivatized using the procedure outlined above. Instrumentation and Operating Conditions. A Shimadzu (Kyoto, Japan) prominence ultrafast liquid chromatography (UFLC) system consisting of two LC-20AD pumps, a highpressure gradient unit, a DGU-20A3 online degasser, a SIL20AC autosampler, and a CTO-20AC column oven was used. An autosampler was used to make 10 μL injections. A FluoroSep-RP Propyl column (100 mm × 2.1 mm i.d., particle size 5 μm, ES industry, West Berlin, NJ, U.S.A.) was used. Solvent A (methanol/water/TFA = 90:10:0.05, v/v) and solvent B (TFE/formic acid = 100:0.1, v/v) were used as the mobile phases for the gradient elution (gradient curve: 0−3 min, 0% B; 3−10 min, linear change from 0−60% B; 10−10.01 min, linear change from 60−0% B; run-time, 15 min). The flow rate was set at 0.3 mL/min. The column oven temperature was set at 40 °C. The effluent from the LC column was directly introduced into the ion source of the mass spectrometer without splitting. For comparison, other fluorous stationary phase columns, such as Fluophase RP (100 mm × 2.1 mm i.d., particle size 5 μm, Thermo Fisher Scientific, San Jose, CA, U.S.A.), Fluofix-II 120E (150 mm × 2.0 mm i.d., particle size 5 μm, Wako Pure Chemicals), and FluoroSep-RP Octyl (100 mm × 2.1 mm i.d., particle size 5 μm, ES industry), were used as analytical columns to assess how their performance differed from the FluoroSep-RP Propyl column. An API 4000 Qtrap tandem mass spectrometer (AB Sciex, Concord, Ontario, Canada) was operated in the positive ESI mode. The following operating conditions were used: an ESI capillary voltage of 5500 V, a source temperature of 650 °C, a curtain gas of 10 (arbitrary units), an ion source gas 1 pressure of 60 (arbitrary units), and an ion source gas 2 setting of 80 (arbitrary units). For multiple reaction monitoring (MRM) mode, the precursor ions, product ions, declustering potential (DP), collision-induced dissociation energies (CE), entrance potential (EP), and collision cell exit potential (CXP) of the fluorous-labeled derivatives are shown in Table 1. Preparation of Calibration Standards. Stock solutions of biogenic amine standards (1 mM) were prepared by dissolving an appropriate amount of each compound in 0.1 M hydrochloric acid. Calibration standards at concentrations of 0.01, 0.05, 0.1, 0.5, and 1 nM for all analytes were prepared by diluting the stock solution with 0.1 M hydrochloric acid to the required concentrations immediately before use. These standards were derivatized with PFUA using the procedure described

Table 1. MRM Conditions for the Derivatives of Biogenic Amines

DA NE 3-MT NM Tyr 5-HT Trp 5-MT His

precursor ion (m/z)

product ion (m/z)

DP (V)

CE (eV)

EP (V)

CXP (V)

1074 1090 1088 1104 1058 1097 1081 1111 1032

137 1072 151 1086 121 950 950 950 950

186 161 161 96 196 126 141 131 191

85 53 87 51 103 69 59 55 81

10 10 10 10 10 10 10 10 10

8 28 12 54 10 44 20 22 46

above and were injected onto the LC−MS/MS system by the autosampler. Peak areas used for the quantification of the biogenic amines were integrated automatically. Analyte concentrations in plasma were calculated using the calibration curves generated from the peak areas of each analyte. The interday precision of the method was estimated using standard solutions (0.01, 0.1, and 1 nM) measured six times each day for 6 days. The LOD was defined as the concentration that produced a signal-to-noise ratio (S/N) higher than 3. Matrix Effects Evaluation. To evaluate the feasibility of our strategy to overcome matrix-induced effects, the method was validated using the approach reported by Matuszewski et al.16 First, we compared the response from standard derivatized amines (nonmatrix samples) and with those spiked at the same levels into extracts of human plasma (matrix samples) to assess the matrix effects (ME). This was achieved by mixing each derivatized standard solution (0.01, 0.1, and 1 nM) with either equal volumes of nonderivatized human plasma previously extracted with isopropyl alcohol (matrix samples) or with an equivalent of 70% isopropyl alcohol (nonmatrix samples). These solutions were analyzed by LC−MS/MS, and the ME were calculated using eq 1: ME = 100 × response in matrix sample /response in nonmatrix sample

(1)

Next, the extraction recovery (ER) was determined by comparing the response of standard solutions spiked at the same levels into human plasma before and after extraction. One set of samples was prepared by combining standard solutions at three concentration levels (0.01, 0.1, and 1 pmol/mL) with human plasma followed by extraction and derivatization, and a second set was prepared by combining the same levels of derivatized standard solutions with previously extracted human plasma. The ER was calculated using eq 2: ER = 100 × (response in plasma spiked before extraction − response in nonspiked plasma)/response in plasma

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spiked after extraction

(2)

RESULTS AND DISCUSSION Evaluation of Fluorous Separation. One of the most effective approaches to eliminating matrix-induced effects in LC−MS/MS analysis is to ensure sufficient analyte separation from endogenous components in biological matrixes. Our fluorous-labeling strategy created a chromatographic handle specific to the analytes that allowed us to achieve excellent

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Figure 2. ESI-MS/MS spectra of the binary fluorous-labeled standard amines: (a) DA, (b) NE, (c) 3-MT, (d) NM, (e) Tyr, (f) 5-HT, (g) Trp, (h) 5-MT, and (i) His. Precursor ions are (a) m/z 1074, (b) m/z 1090, (c) m/z 1088, (d) m/z 1104, (e) m/z 1058, (f) m/z 1097, (g) m/z 1081, (h) m/ z 1111, and (i) m/z 1032. The collision-induced dissociation energy was 60 eV for all derivatives.

(nonfluorous) but also the excess derivatization reagent and derivatized secondary amines, which were only monoperfluoroalkylated. On this basis, they were selectively retained on the LC column and separated from sources of matrix interference prior to MS/MS analysis. This approach requires the use of a reductant to drive the reductive amination reaction. Sodium cyanoborohydride is most commonly utilized for this purpose but is highly toxic. As an alternative, 2-PB has been recently employed as a reductant in place of sodium cyanoborohydride because it is nontoxic, exhibits high selectivity for iminium reduction, and is highly stable.33,34 Therefore, we chose to use 2-PB as the reductant for reductive amination in the present study. To optimize the derivatization reaction, we tested various 2-PB concentrations ranging from 0.2 to 2.0 M under a fixed PFUA concentration (1.5 M) and found that the peak intensities of all derivatives increased with increasing concentrations of 2-PB up to 1.2 M. On the basis of this result, we chose to use 1.5 M 2-PB for the derivatization, and all subsequent optimization studies were carried out using this condition. The concentration of PFUA was also found to affect the peak intensities of the derivatives and was optimized as well. By varying the concentrations of PFUA from 0.01 to 2.5 M, 2.0 M PFUA was found to produce the maximum intensities of most of the target analytes and was selected as the optimal concentration. The derivatization was carried out at room temperature, as higher temperatures were found to slightly decrease the peak intensities of the derivatives. To optimize the reaction time, the progress of the reaction was assessed over the course of 60 min. It was found that the peak intensities reached their maximum values at 15 min, after which they remained

matrix−analyte separation. To characterize the specificity of the separation mechanism, we assayed primary amines derivatized with PFUA or with n-undecylaldehyde (which has the same carbon number as PFUA) under our LC conditions. The results of this fluorous separation study are shown in the Supporting Information Figure S1. The binary fluorous-labeled amines were retained more strongly than the nonfluorous alkyl aldehyde-labeled amines, indicating that the binary fluorous derivatives were selectively retained on the fluorous LC column on the basis of fluorophilicity and not hydrophobicity.29,30 Furthermore, we examined the retention of diphenylamine, which was used to consume excess reagent and reacted with a single PFUA label. Despite the moderate fluorophilicity of the monofluorous-labeled diphenylamine, it was poorly retained on the fluorous LC column (Supporting Information Figure S1a) under our LC conditions, which includes the use of extremely high fractions of organic solvent as mobile phase. Through these experiments, we demonstrated that binary fluorouslabeled amines were selectively retained on the column, enabling excellent separation of analytes from matrix-related species and ultimately leading to the elimination of matrixinduced effects in LC−MS/MS analysis. Derivatization of Biogenic Amines. Reductive amination is a simple and specific reaction between an amine and aldehyde that can transform either primary or secondary amines into the corresponding tertiary amines by the introduction of two or one alkyl groups, respectively. In this study, the target biogenic amines were diperfluoroalkylated with PFUA via reductive amination, giving them a distinctly greater fluorophilicity than not only the species in the biological matrix 8410

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Figure 3. MRM chromatograms of the binary fluorous-labeled standard amines (0.5 nM each): (a) DA, (b) NE, (c) 3-MT, (d) NM, (e) Tyr, (f) 5HT, (g) Trp, (h) 5-MT, and (i) His.

fragmented, and the resulting product ions were determined by scanning the third quadrupole. The ESI-MS/MS spectra of the protonated derivatives are shown in Figure 2. Besides common fragment peaks at m/z 950 and/or 938, some characteristic fragments derived from each intact structure corresponding to cleavage products of the form [M + H − NH(C3H6C8F17)2]+, e.g., m/z 137 for DA, m/z 151 for 3-MT, m/z 121 for Tyr, m/z 160 for 5-HT, m/z 144 for Trp, m/z 174 for 5-MT, and m/z 95 for His, were obtained for all derivatives except for NE and NM. In the MS/MS spectra of both of NE and NM derivatives, the protonated ions minus water [M + H − H2O]+ were observed as the dominant fragments. In order to maximize sensitivity in the detection of the target amines, the most intense precursor−product ion transitions and their related parameters were selected as the MRM quantification transitions (Table 1). Fluorous LC Conditions. The diperfluoroalkylated amines were very strongly retained on the fluorous LC column in the presence of hydrophobic mobile phase. In preliminary studies, a weakly acidic mixture of water, methanol, and acetonitrile was tested as mobile phase but was found to produce overly long retention times and broadening of the analyte peak shapes. We found that using a polar fluorous alcohol, such as TFE, as a coeluent in the mobile phase allowed us to obtain good

constant. On the basis of all these results, the use of 1.5 M 2-PB and 2.0 M PFUA with a reaction time of 15 min at room temperature was chosen as optimal conditions for the dialkylation of biogenic amines through reductive amination. The derivatives were found to be stable for at least 48 h at 4 °C when stored in the dark. MRM Conditions in ESI-MS/MS for Derivatized Amines. In general, LC−ESI-MS is most effective for hydrophobic compounds because of their increased retention in reversed-phase LC and their ionization efficiency. Hydrophobic ions preferentially locate to the electrospray droplet surface layer and enter the gas phase more readily when the solvent evaporates, making it difficult to achieve sufficient sensitivity for the detection of hydrophilic compounds by LC− ESI-MS, such as the biogenic amines targeted in this study. Although we selected our derivatization strategy in order to achieve good separation of analytes from interfering species, the hydrophobicity of the perfluoroalkyl-containing derivatization reagent also served to enhance the ESI response of biogenic amines. The ionization conditions for analytes in ESI mode were optimized using derivatized standard samples. All derivatives were found to be ionizable in positive ionization mode, forming protonated ions [M + H]+ in the first quadrupole. These were selected as precursor ions and further 8411

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retention times and sharp peak shapes for the derivatives. By using a fluorous solvent, we could easily control the elution of perfluorinated compounds from the fluorinated stationary phase. Fluorous solvents are typically very nonpolar and usually cannot be used in reversed-phase LC or ESI-MS. However, TFE is an exception and has been utilized as a controllable solvent for the elution of fluorinated compounds in fluorousphase and reversed-phase LC.35,36 To optimize the mobile phase composition, we varied the concentration of TFE in the mobile phase used for gradient elution and assessed the resulting analyte retention times, finding that increasing concentrations of TFE in the mobile phase shortened the retention times of derivatives as anticipated. On the basis of these findings, we used one mobile phase without TFE (solvent A) and one with TFE (solvent B) to carry out a gradient elution that produced complete separation of the derivatized analytes from nonfluorous and monofluorous components, good peak shapes, and appropriate retention times. Additionally, the fluorinated stationary phase did not separate based on fluorophilicity alone but also displayed normal phase behavior, especially for basic compounds.35,37,38 This phenomenon most likely arose from the significant number of silanol residues in silica gel-based fluorinated phases. As expected, the use of high percentages of organic solvent in the mobile phase dramatically increased the retention of mono- and binary fluorous labeled amines on the fluorous LC column, accompanied by an increase in peak broadening (data not shown). To correct for this, TFA was added to solvent A as a volatile ion-pair reagent to improve the behavior of basic derivatives on the fluorous LC column. Formic acid was added to solvent B to prevent signal suppression in the ESI source from TFA.39,40 The final compositions of solvents A and B were water/methanol/TFA and TFE/formic acid, respectively, used at a flow rate of 0.3 mL/min in the gradient elution in this study. Under these conditions, all derivatives could be eluted within 10 min (Figure 3). Furthermore, we examined the retention behavior of binary fluorous-labeled amines on several fluorous columns, such as the Fluophase RP (100 mm × 2.1 mm i.d., particle size 5 μm), Fluofix-II 120E (150 mm × 2.0 mm i.d., particle size 5 μm), and FluoroSep-RP Octyl (100 mm × 2.1 mm i.d., particle size 5 μm) columns, for comparison. Although all the columns tested were able to strongly retain the binary fluorous derivatives on the basis of fluorophilicity and not hydrophobicity, the shapes and sharpness of the analyte peaks on the FluoroSep-RP Propyl column were better than those produced using the other columns under our optimized LC conditions, especially in derivative of His (data not shown). Therefore, the FluoroSepRP Propyl column was used for this study. Analysis of Standards. We investigated the sensitivity, linearity, and repeatability of the determination of biogenic amines using fluorous derivatization in tandem with LC−MS/ MS analysis using standard solutions for method validation. The relationship between the derivative peak areas and the concentration of amines in standard solutions was linear over a concentration range of 0.01−1 nM. The correlation coefficients of the calibration curves, the interday precision values, and the LODs for amines detected in MRM mode are shown in Table 2. All correlation coefficients were greater than 0.9978. The interday precision values were established by repetitive analysis (n = 6) using standard solutions at concentrations of 0.01, 0.1, and 1 nM; the relative standard deviations (RSDs) were within 7.4%. The LODs of the target biogenic amines using our

Table 2. Linearity, Limit of Detection, and Repeatability RSD (%, n = 6)c a

DA NE 3-MT NM Tyr 5-HT Trp 5-MT His

linearity (r)

LOD (amol)

0.9993 0.9985 0.9999 0.9996 0.9983 0.9978 0.9997 0.9984 0.9991

26 7.8 21 18 15 9.6 10 10 16

b

0.01 nM

0.1 nM

1 nM

2.8 4.0 3.5 4.6 4.7 3.9 5.0 7.4 3.8

5.9 5.3 4.8 3.4 5.1 1.0 3.2 2.7 5.2

2.7 1.4 3.0 6.0 2.4 1.9 2.2 2.2 1.7

a

Correlation coefficient of calibration curves of derivatives in the range from 0.01 to 1 nM. bDefined as a signal-to-noise ratio greater than 3. c Relative standard deviations of peak areas of derivatized amines.

optimized conditions were 7.8−26 amol on column. These obtained LODs of biogenic amines were far lower than those for a previously reported LC−MS/MS method without derivatization,41 and moreover, they were nearly equal to or lower than those obtained using previous LC−MS/MS methods with detection-oriented derivatization technique.7,12 Application to Human Plasma Analysis. To demonstrate the utility of this method for eliminating matrix-induced effects in LC−MS/MS analysis, it was applied to the analysis of several human plasma samples. In the analysis of plasma, phospholipids are known to be the main endogenous component that causes matrix-induced effects, as they have all the desirable properties for ESI-MS analysis including high ionic potential and hydrophobicity.24,42,43 Therefore, phospholipids in plasma samples were specifically monitored in order to evaluate the effectiveness of the fluorous LC separation to eliminate this source of interference. The precursor ions were scanned when a peak at m/z 184 was detected as a product ion to monitor the phospholipids in plasma, as the trimethylammonium-ethyl phosphate ion (m/z 184) is typically used as the common fragment ion of phospholipids in MS/MS analysis (Figure 4). It was found that phospholipids were not retained on the fluorous-phase LC column, meaning that the derivatized analytes could be completely separated from this interfering species endogenous to biological matrix. Furthermore, to confirm that our method could eliminate matrix-induced effects, human plasma spiked with several different levels of the target amines was analyzed. As shown in Table 3, the ME and ER values obtained for human plasma samples were in the ranges of 95.9−106% and 95.9−108%, respectively, for all levels examined. In contrast, in the previously reported LC−MS/MS method with derivatization technique, matrix-induced effects were observed to be more than 100% ± 10% for the analysis of biogenic amines in human plasma.12 These results demonstrate that this LC−MS/MS method in conjunction with fluorous derivatization enables the accurate and precise analysis of biogenic amines in biological samples by eliminating matrixinduced effects from endogenous components Several native human plasma samples were also analyzed using this method. The samples were collected from healthy volunteers, six men and three women. Trace amounts of all target amines were detected in these plasma samples, which was possible because of the high sensitivity of the method (Figure 4). The concentration ranges of the amines in human plasma were as follows: DA 0.10−0.95, NE 0.10−1.1, 3-MT 0.02−0.50, NM 0.02−0.43, Tyr 0.42−4.2, 5-HT 8.9−57, Trp 1.2−3.2, 58412

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Figure 4. Chromatograms obtained from the analysis of human plasma sample: (a) DA (0.10 pmol/mL plasma), (b) NE (1.1 pmol/mL plasma), (c) 3-MT (0.28 pmol/mL plasma), (d) NM (0.43 pmol/mL plasma), (e) Tyr (0.81 pmol/mL plasma), (f) 5-HT (20 pmol/mL plasma), (g) Trp (1.9 pmol/mL plasma), (h) 5-MT (0.04 pmol/mL plasma), (i) His (20 pmol/mL plasma), and (j) phospholipid. The derivatized amines and the phospholipids were monitored in MRM mode and precursor ion scan mode using the product ion m/z 184, respectively.

Table 3. Evaluation of Matrix Effects Using the Described Method ME

ER

(mean ± SD %, n = 3) 0.01 nM DA NE 3-MT NM Tyr 5-HT Trp 5-MT His

101 100 100 100 103 97.1 96.5 100 103

± ± ± ± ± ± ± ± ±

5.4 6.6 3.6 2.3 1.1 3.8 0.9 3.9 1.1

0.1 nM 102 99.4 99.2 106 100 102 103 96.2 103

± ± ± ± ± ± ± ± ±

8.2 4.9 6.7 2.3 4.4 1.2 6.1 5.4 8.2

(mean ± SD %, n = 3) l nM 97.5 98.3 101 97.2 100 101 102 96.7 95.9

± ± ± ± ± ± ± ± ±

0.01 pmol/mL plasma

5.1 4.2 1.6 6.7 7.1 4.6 8.1 0.6 4.7

106 102 101 102 99.5 101 102 102 108

2.5 2.6 5.4 3.7 2.0 1.7 4.9 4.0 1.2

0.1 pmol/mL plasma 102 100 101 99.9 98.2 102 103 102 101

± ± ± ± ± ± ± ± ±

5.7 7.9 2.1 7.1 4.0 2.4 2.9 3.2 4.3

1 pmol/mL plasma 95.9 99.3 104 100 98.6 97.0 99.1 104 98.8

± ± ± ± ± ± ± ± ±

4.6 0.9 3.9 4.9 4.2 1.5 2.8 1.3 2.1

the fluorous derivatization of analytes with PFUA via reductive amination followed by fluorinated stationary phase LC−MS/ MS analysis. The derivatized biogenic amines were diperfluoroalkylated and as a result were very strongly retained on a fluorous LC column. Subsequent elution of analytes was controlled through the amount of TFE used in the mobile phase. This derivatization method enabled the efficient

MT