Stepwise fluorometric determination of primary and secondary amines

Gas chromatography/mass spectrometry determination of water-soluble primary amines as their pentafluorobenzaldehyde imines. Michael J. Avery and Grego...
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Anal. Chem. 1984, 56,919-922

Stepwise Fluorometric Determination of Primary and Secondary Amines by Liquid Chromatography after Derivatization with 2-Methoxy-2,4-diphenyl-3(2H)-furanone Hiroshi Nakamura,* Kazuko Takagi, and Zenzo Tamura' Department of Analytical Chemistry, Faculty of Pharmaceutical Sciences, University of Tokyo, 7-3-1,Hongo, Bunkyo-ku, Tokyo 113,J a p a n

Reiko Yoda and Yuichi Yamamoto2 Laboratory for Medicinal Chemistry, Kyoritsu College of Pharmacy, 1-5-30,Shibakoen, Minato-ku, Tokyo 105,Japan

A high-performance llquld chromatographic (HPLC) method was developed for the stepwise fluorometric determlnatlon of prlmary and secondary amines. Amines were reacted wlth 2-methoxy-2,4-dlphenyl-3( 2H)-furanone (MDPF) at pH 9.6 and 20 OC for 30 mln to produce fluorescent pyrrolinones (FI') from primary amines and nonfluorescent aminodlenones (FII') from secondary amlnes. The MDPF-adducts of amlnes were separated on a reversed-phase CI8 (TSK LS-410 K) column wlth a mlxture of methanol and 50 mM phosphate buffer (pH 7.0) (70:30). After the detectlon of FI' wlth the first fluorescence monltor (A,x 360 nm, A,, >405 nm), the eluate was mlxed wlth 12 M eihanolamlne hydrochloride (pH 10.5) to convert F I I' to fluorescent MDPF-ethanolamlne whlch was detected with the second fluorescence monitor (A,, 390 nm, A, 480 nm). The present method permits the determlnatlon of 3 pmol of lower n-alkylamlnes and 50 pmol of lower di-n -aikylamlnes. The relative standard deviations were 2.3-2.7% for 50 pmoi of the n-alkyiamlnes and 2.9-3.4% for 1 nmol of the dl-n-alkylamlnes,


Scheme I







Several high-performance liquid chromatographic (HPLC) methods are available for the simultaneous determination of primary and secondary amines. They may be classified into two types from the viewpoint of strategies employed. One involves the use of labeling agents such as 1-(dimethylamino)naphthalene-5-sulfonylchloride (DNS-C1) (1-31,7 chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-C1) (4-6),and 7-fluoro-4-nitrobenzo-2-oxa-l,3-diazole (NBD-F) (7-9)which derivatize both primary and secondary amines. The other is based on the use of the o-phthalaldehyde/2-mercaptoethanol reagent (lo),which is selective to primary amines, after the postcolumn conversion of secondary amines to primary amines with oxidizing agents such as sodium hypochlorite (11-13)and chloramine-T (14).However, none of these methods permits distinguishing directly whether the amines of question are primary or secondary. 2-Methoxy-2,4-diphenyl-3(2H)-furanone (MDPF) is known to react with primary and secondary amines to give fluorescent pyrrolinones (FI') and nonfluorescent aminodienones (FII'), respectively (15). Excess MDPF is rapidly hydrolyzed to nonfluorescent product (HP') under the reaction conditions for amines (16). In a previous paper we developed the method for the fluorometric determination of secondary amines based on our finding that FII' was converted by the


'Present address: Keio University Hospital Pharmacy, 35, Shinanomachi, Shinjuku-ku, Tokyo 160, Japan. 'Deceased September 6, 1983. 0003-2700/84/0356-0919$01.50/0

reaction with primary amines to FI'. As an extension of the work, a novel HPLC method which provides the stepwise detection of primary and secondary amines in a single injection has been developed in the present investigation, based on the reactions depicted in Scheme I. The present method consists of the separation of MDPF-labeled amines (FI' and FII'), selective detection of FI' with the first fluorescence monitor, and the successive detection of FII' with the second fluorescence monitor after the postcolumn conversion to FI' with ethanolamine. n-Alkylamines and di-n-alkylamines were selected as models for primary and secondary amines, respectively.

EXPERIMENTAL SECTION Materials. MDPF, n-alkylamines, and di-n-alkylamines were purchased from Tokyo Kasei (Tokyo, Japan). Other chemicals and solvents used were of the highest purity commercially available. Preparation of Stock Solutions of Amines. Ten millimolar stock solutions of the amines were prepared with distilled water whenever possible or with acetonitrile. Distilled water was used to prepare dilute solutions. Standard Labeling Procedure for Primary and Secondary Amines. A 100-pLaqueous sample solution was transferred to a 12 X 75 mm glass test tube and 100 p L of 50 mM sodium borate buffer at pH 9.6 was added. While the test tube was vigorously agitated on a vortex-type mixer, 100 WLof 10 mM MDPF solution in acetonitrile was added rapidly. The test tube was further agitated for 10 s and then placed in a water bath equilibrated at 20 "C. After 30 min, the aliquot was injected to the HPLC system. 0 1984 American Chemical Society









EA -









Figure 1. HPLC flow diagram for the stepwlse fluorometric detection of primary and secondary amines: P, and P,, pumps; D, and D,, fluorescence detectors; EA, ethanolamine reagent.

Preparation of Postcolumn Reagent. Ethanolamine was titrated with concentrated hydrochloric acid to pH 10.5. HPLC Apparatus. The flow diagram for the HPLC postcolumn derivatization of amines is shown in Figure 1. All tubing, coils, and loops used were made of stainless steel (0.5 mm i.d. x inch 0.d.). The solvent was delivered through a Mini-micro pump (Type KHD-16; Kyowa Seimitsu, Tokyo, Japan) at a flow rate of 0.45 mL/min. A reversed-phase column (TSK LS-410 K, 5 pm, 4 mm i.d. X 30 cm; Toyo Soda, Tokyo, Japan) was used at ambient temperature. In-stream injection of samples was performed with a 10-pL Hamilton syringe through a line sample injector (Type KLS-3T Kyowa Seimitsu) which was connected to a six-way valve (Type KMH-6V; Kyowa Seimitsu) attached with a 55-pL loop. The eluate was introduced to a 14-pL quartz flow cell in a fluorescence detector (Type FLD-1; Shimadzu Seisakusho, Kyoto, Japan) equipped with a coated low-pressure mercury lamp emitting light at 300-400 nm (maximum intensity at 360 nm) and an EM-3 secondary filter which cuts off light at wavelengths shorter than 405 nm. The fluorescence intensity was recorded with a two-pen recorder (Type R-12; Shimadzu Seisakusho). The outlet of the flow cell was connected to a 48cm length of tubing and the eluate was mixed in a three-way tee with the postcolumn reagent delivered at a flow rate of 0.25 mL/min with a Mini-micro pump (Type KHD-16; Kyowa Seimitsu). The outlet of the tee was connected to a 3-m heating coil maintained at 60 "C in a water bath and was connected to a 50-cm cooling coil which was immersed in an ice bath. The cooling of the reaction mixture was necessary to enhance the fluorescence intensity. The end of the cooling coil was introduced to a 12-pL quartz flow cell in a fluorescence spectromonitor (Type RF-530; Shimadzu Seisakusho) equipped with a 75-W xenon lamp. The fluorescence intensity was monitored at 480 nm with 390-nm excitation and recorded with the two-pen recorder. The outlet of the flow cell was connected to a 10-m back pressure coil whose end was immersed in the ethanolamine trap filled with a phosphoric acid solution.

RESULTS AND DISCUSSION Labeling of Amines with MDPF. Since the fluorescence intensity of the blank was almost negligible, the optimization of the labeling reaction of primary amines with MDPF, which produced fluorescent FI', was examined with a flow injection technique. In contrast, the reactions between secondary amines and MDPF were followed by separating the nonfluorescent FII' from interfering MDPF and HP' with the condition described below and by converting FII' to FI' with ethanolamine. The reactions of n-alkylamines with MDPF proceeded above pH 8.5 and the peak height of FII' was almost constant between pH 9 and pH 10.5. Similar results were obtained with diethylamine, di-n-pentylamine, and di-n-hexylamine. When the amines were reacted a t pH 9.6 and 20 "C for 5-10 min with varying concentration of MDPF, the fluorescence intensities reached plateaus with MDPF concentration higher than 5 mM for n-alkylamines and 10 mM for di-n-alkylamines. The labeling reaction carried out a t pH 9.6 using 10 mM MDPF was influenced by reaction temperature, especially in



Figure 2. Effect of temperature on the MDPF labeling reactions.

Samples of 0.3 mM n-alkylamine (a)and 5 mM each of dimethylamine

and dl-n-pentylamine(b) were treated with the standard labeling procedure except that the temperature was variable and that the reaction time was 5 rnin (a) or 10 mln (b).





Flgure 3. Effect of temperature on the stability of FII'. A sample of 1 mM dimethylamine or 0.5 mM dl-n-pentylamine was treated with the standard labeling procedure. A 100-pL allquot of the reaction mixture was added to 100 pL of 0.2 M sodlum borate buffer (pH 9.6) and incubated at various temperatures for 30 min.

the case of secondary amines (Figure 21, because both the hydrolysis of MDPF and the degradation of FII' were accelerated at elevated temperatures. When conducted a t pH 9.6 and 20 "C using 10 mM MDPF, the reactions of n-alkylamines and di-n-alkylamines with MDPF were completed within 15 min. Stability of FI' a n d FII'. The MDPF adducts of n-alkylamines (FI') were stable at room temperature for at least 2 h. However, those of di-n-alkylamines (FII') were inclined to be gradually decomposed. Therefore, the effect of pH on the stability of MDPF-adducts of di-n-alkylamines was examined by allowing the aliquot of the reaction mixture to stand a t 20 "C for 30 min in the large excess of media with various pHs. The stability of FII' was found to be maximal at pH 9-10. When kept at pH 9.6 for 30 min, FII' was stable at 0-20 "C; however, the decomposition of FII' was observed above 20 "C with increasing temperature (Figure 3). Conversion of FII' t o FI'. When the MDPF adducts of dimethylamine and di-n-pentylamine were reacted at 70 "C with 5 M ethanolamine solutions adjusted to various pHs with concentrated hydrochloric acid solution, the fluorescence intensities due to the formation of MDPF-ethanolamine were scarcely observed below pH 8 and were maximal at pH 10. Similarly, the optimal pH for di-n-hexylamine was found to be 11. While the fluorescence intensity that originated from dimethylamine reached a plateau a t pH 10.5 and 70 "C with ca. 2 M ethanolamine hydrochloride, that from di-n-pentylamine continued to increase even with 1 2 M ethanolamine hydrochloride. On the other hand, the conversion reactions







TIME I min

Figure 4. Separation of the MDPF derivatives of n-alkylamine homologues (Cl-C8). The MDPF-adduct corresponding to 400 pmol of n-alkylamine was injected: column, TSK LS-410 K (4 mm i.d. X 30 cm); eluent, methanol-50 mM phosphate buffer at pH 7.0 (70:30).

were not so influenced by the temperature between 20 "C and 95 "C, it being maximal at around 60 "C with a broad profile over the temperature range. As to the effect of coil length on the conversion reactions, the coil length of 3 m, corresponding to 50.5 s of the residence time, seemed to be optimal. On the basis of the above findings, the conditions for the postcolumn conversion of FII' to FI' were established as described in Experimental Section. Separation of MDPF-Labeled Amines. Six kinds of MDPF-labeled n-alkylamine homologues from methylamine (C,) to n-hexylamine (C,) were separated in 40 min on a reversed-phase C18-bondedsilica gel column (TSK LS-410 K) by an isocratic elution with methanol-50 mM phosphate buffer at pH 7.0 (70:30) (Figure 4). Under the same conditions, four MDPF derivatives of di-n-alkylamines from dimethylamine (C,') to di-n-butylamine (Cd) were also eluted in ca. 40 min. To separate the MDPF derivatives of di-npentylamine and di-n-hexylamine in a practical time, the isocratic elution with 90% methanol was effective. Although the chromatographic efficiency was rather poor, no further effect was made for the improvement of it since the focus of the present investigation was in the chemistries of detection. Unlike the case of detection of the primary amines, the chromatograms obtained with the second fluorescence detector (D2in Figure 1)always included interfering peaks due to the unhydrolyzed MDPF and HP'. Figure 5 shows an example of the stepwise fluorometric detection using MDPF derivatives of selected primary and secondary amines. The calibration curves constructed by plotting the peak height against the amount of amine passed through the origin and were linear for up to 4 nmol of amine injected. When the isocratic elution in Figure 4 was employed, the limits of determination were 3 pmol for methylamine, ethylamine, n-propylamine, and n-butylamine and 50 pmol for dimethylamine, diethylamine, di-n-propylamine, and di-n-butylamine. The detection limits (SIN = 2) of the lower n-alkylamines were 0.5 pmol and those of the lower di-n-alkylamines were 10 pmol. The precision of the method was evaluated by analyzing the mixture of methylamine, diethylamine, n-pentylamine, and di-n-butylamine five times. The relative standard deviations were 2.3% for 50 pmol of methylamine, 2.9% for 1nmol of diethylamine, 2.7% for 50 pmol of n-pentylamine, and 3.4% for 1nmol of di-n-butylamine. Among many fluorogenic reagents for amines, only MDPF and fluorescamine (FLA) possess unique characteristics. Although the reactivity of FLA toward amines is quite similar to that of MDPF (18), a similar attempt with FLA to the present work had been unsuccessful because of the unstability







Figure 5. Stepwise fluorometric detection of primary and secondary amines as their MDPF derivatives. The MDPF adducts corresponding to 200 pmol each of methylamine (C,) and n-pentylamine (C5), 2 nmol of diethylamine (CSr),and 1 nmoi each of di-n-propylamine(CSr)and di-n-butylamine (Ci) were injected. Conditions are given in Figure 4.

of FLA adducts of secondary amines (19). FI' and FII' are much more stable than the corresponding FLA primary amines (FI) and FLA secondary amines (FII) owing to the lack of a carboxyl group in their molecules as discussed previously (17). The detection sensitivity of the present method for secondary amines is less than one-tenth that for primary amines, which is considered to be due to both the decomposition of FII' and the incomplete conversion of FII' to MDPF-ethanolamine. Unhydrolyzed MDPF and the hydrolysis product of MDPF (HP') interfered in the detection of lower secondary amines of minute amounts. To overcome this problem, the performance of the labeling reaction with lower concentration of the MDPF reagent, at lower temperature for a longer time, might be effective since the fluorescence intensity of HP' ww less than 1% of that of MDPF itself in the conversion reaction. The present method will be useful for distinguishing primary and secondary amines in unknown samples. The analysis of polyamine metabolites in biological materials by the present method is now in progress. Registry No. MDPF, 50632-57-0; methylamine, 74-89-5; ethylamine, 75-04-7;propylamine, 107-10-8;n-butylamine, 10973-9; n-pentylamine, 110-58-7; n-hexylamine, 111-26-2; dimethylamine, 124-40-3;diethylamine, 109-89-7;dipropylamine, 142-84-7;dibutylamine, 111-92-2;di-n-pentylamine, 2050-92-2; di-n-hexylamine,143-16-8;ethanolaminehydrochloride, 2002-24-6.

LITERATURE CITED (1) Yamabe, T.; Takai, N.; Nakamura, H. J . Chromatogr. 1975, 104, 359-365. (2) Bayer, E.; Grom, E.; Kaltenegger, B.; Uhmann, R. Anal. Chem. 1978, 48, 1101-1109. (3) Werkhoven-Goewie, C. E.; Brinkman, U. A. Th.; Frei. R. W. Anal Chlm. Acta 1980, 114, 147-154. (4) Wolfram, J. H.; Feinberg, J. I.; Doerr, R. C.; Fiddler, W. J . Chromatogr. 1977, 132, 37-43. (5) Ross, M. Clln. Chlm. Acta 1978, 8 3 , 273-277. (6) Ahnoff, M.: Grundevik. I . ; Arfwidsson, A,; Fonselius, J.; Persson, 5.-A. Anal. Chem. 1981, 5 3 , 405-469. (7) Watanabe, Y.; Imai, K. Anal. Blochem. 1981, 116, 471-472. (8) Watanabe, Y.; Imai, K. J . Chromatogr. 1982, 239, 723-732. (9) Imai, K.; Watanabe, Y.; Toyo'oka, T Chromatographia 1982, 16. -2 14-2 . . - 15 .-. (10) Benson, J. R.; Hare, P. E. R o c . Natl. Acad. Sci. U . S . A . 1975, 72, 619-622. (11) Bohlen, P.; Mellet, M. Anal. BlOCh8m. 1979, 9 4 , 313-321


Anal. Chern. 1984, 56, 922-925

(12) Ishida, Y.; FuJlta, T.; Asai, K. J. Chromatogr. 1981, 204, 143-148. (13) Himuro, A.; Nakamura, H.; Tamura, 2 . J . Chromatogr. IS83, 264, 423-433. (14) Drescher. D.;Lee, K. S. Anal. Biochem. 1978, 8 4 , 559-569. (15) Weigele, M.; Tengi, J. P.; DeBernardo, S.; CzaJkowski, R.; Leimgruber, W. J. Org. Chem. 1976, 4 1 , 388-389. (16) Weigeie, M.; DeBernardo, S.; Leimgruber, W.; Cleeland, R.; Grunberg, E. Blochem. Biophys. Res. Commun. 1973, 5 4 , 899-906.

(17) Nakamura, H.; Tanii, E.; Tamura, 2 . ; Yoda, R.; Yamamoto, Y. Anal. Chem. lS82, 5 4 , 2482-2485. (18) Nakamura, H. DoJn Nyusu lS80, No. 2 0 , 1-12. (19) Nakamura, H.; Tamura, 2 . A n d . Chem. 1980, 5 2 , 2087-2092.

for review December 12, lg83* Accepted January

31, 1984.

Thermal Lens Absorption Measurements on Small Volume Samples C. A. Carter and J. M. Harris* Department of Chemistry, University of Utah, Salt Lake City, Utah 84112

Thermal lens calorimetry has several attributes that make It suitable for small volume detection, Including high sensitivity and use of focused laser excitation. This work considers the fundamental trade-off between detection volume and sensltlvity, which Is related to divergence of the focused beam. A criterion for minimum sample volume for a given path length Is derived. Sources of noise which depend on how tightly the laser Is focused through the sample are considered. Sensltlvlty and precision of thermal lens measurements made on 1 mm path length, smaller volume and 1 cm path length, larger volume samples are compared.

Recent developments in packed microbore ( I ) and open tubular (2)liquid chromatography and flow injection analysis (3) are demanding ever lower dead-volume detectors. Laser-based optical detectors have been described (4-6)as being uniquely suited to this task due to the spatial coherence of laser radiation which allows focusing of high optical powers onto extremely small areas. Fluorescence methods have received the most attention thus far because of the low detection limits attainable in sample volumes ranging from a few microliters to picoliters (5, 7-9). Fluorescence detection is restricted, however, to molecules having significant quantum yields or which can be derivatized with a fluorescent tag. Thermal lens calorimetry ( 1 0 , l l )is an emerging technique for the sensitive detection of molecules which are not strongly fluorescent. Low detection limits have been demonstrated with static (12-21) and flowing (22-24) samples in 1.0 cm path length cells. A recent demonstration of thermal lens measurements with a 1.0 mm path length, 0.5-pL sample cell points out the capability of this method for small volume detection (25). In the present work, we consider the effect of tight beam focusing, required for small volume detection, on the photometric sensitivity and precision of thermal lens measurements. A direct comparison is made of results gathered with instrumentation optimized for 1 mm path length, small volume detection vs. that optimized for 1cm path length, larger volume samples.

THEORY A thermal lens is produced in a sample by the nonradiative loss of energy absorbed from a laser beam. Due to the Gaussian radial intensity distribution of the beam, a greater amount of heat is deposited at the beam center than at the edges, generating a temperature gradient which acts in most

liquids as a diverging lens. Under continuous illumination, the thermal lens reaches a steady-state strength given as the inverse focal length by (26)

2.303P(dn/di")A l/f(m) =



where P and w are the laser power and spot size, respectively, (dn/dT) is the variation of refractive index with temperature, A is the decadic absorbance, and k is the thermal conductivity of the sample. The strength of the thermal lens is generally measured by observing the relative change in the beam-center intensity, h l b c / I b c , in the far field. The intensity change optimizes for weak absorbance measurements when the sample is located 3lI2Zcbeyond a waist in the beam to yield (27,28) AIbc/Ibc

= 2.303EA

+ (2.303EA)2/2


where the enhancement relative to a transmission measurement is given by

E = -P(dn/di")/l.SlXk


and the confocal distance is, Z, = r w : / X , where wo is the spot size of the laser beam at its waist and X is the wavelength. Since the goal of small volume detection is to focus the beam as tightly as possible through the sample, it is interesting to note that the sensitivity predicted by eq 2 and 3 does not depend on the beam spot size. This is somewhat surprising since the strength of the thermal lens increases with decreasing spot size, as eq 1 indicates. The concomitant increase in the divergence angle of the beam with decreasing spot size, 0 = ( X / r w O ) ,exactly cancels the effect of the stronger thermal lens on relative intensity change, hl),,/Ib,. As a result, for a given sample absorbance, reducing the detection volume by decreasing the beam spot size should not affect the sensitivity of the thermal lens measurement. Another aspect of the measurement, the time dependence, is strongly affected by spot size. The characteristic time constant for the formation of a thermal lens is given by (28, 29) t , = w2pCp/2k (4) where p and C, are the density and specific heat of the sample. The spot size of the beam at an optimally positioned sample, coopt, is proportional to the waist spot size, wo, since w2

= w t ( 1 + (2/2,)2)


where Zopt= 3lI2Zcand therefore coopt = 2w0. Consequently,

0 1984 American Chemical Society 0003-2700/84/0356-0922$01.50/0