2380
Anal. Chem. 1986,58,2380-2383
Determination of Triglycerides by High-Performance Liquid Chromatography with Postcolumn Derivatization Yukihiro Kondoh and Satoehi Takano* Tochigi Research Laboratories, Kao Corporation, 2606, Akabane, Ichikai-machi, Tochigi 321 -34,J a p a n
A new postcolumn reactor detector for the analysis of triglycerides with high sensitlvity, hlgh selectlvlty, and molar respondbl#ty has been developed. Triglycerides eluted from a hlgh-performance llquw chromatographic column are hydrolyzed with potasslum hydroxlde, and the resulted glycerin is oxidized to formaldehyde with periodic acid. Then formaldehyde Is reacted with acetyiacetone in the presence of ammonlum acetate to form 3,5-dlacetyi-1,4-dlhydrolutldlne, which is detected at 410 nm. The detector, for example, can detect 0.1 nmol of trliaurln and give a linear working curve between 0.3 and 60 nmd of trllaurln. With this postcolumn reactor detector, trlgiycerides of natural fats and oils are sufficiently separated and quantitatively analyzed with nonaqueous reversed-phase chromatography uslng ODS column. Better separations were demonstrated by using a gradient solvent system.
Recently many studies on the analysis of triglycerides have been reported by means of high-performance liquid chromatography (HPLC). Almost all have used nonaqueous reversed-phase (NARP) chromatography with an octadecyl chemically bonded silica (ODS) column. Dong et al. ( I ) accomplished the high-resolution separations of triglycerides in natural fats and oils such as palm oil and olive oil etc. within 8-16 min. Better separations were demonstrated by using a gradient solvent system (2-7). Many detectors are now commercially available. A refractive index detector (RID) is most frequently used ( I , 8, 9) for the analysis of triglycerides; however, this detector has poor sensitivity and no selectivity. Other detectors such as a UV detector (lo),a flame ionization detector (FID) (2,3), an infrared (IR) detector ( 4 ) ,a mass detector (5,6),a laser light scattering detector (3,and a postcolumn reactor detector (PCRD) (11) are also used for the analysis of triglycerides. Among these detectors, a laser light scattering detector has the highest sensitivity; however, it, unfortunately, lacks selectivity (7). Compton et al. (11)developed a PCRD by using Fluoral-P (12)as a color development reagent for the selective detection of triglycerides and phospholipids. However, the sensitivity was not high enough and satisfactory resolution for the analysis of triglycerides of natural fats and oils could not be achieved because of the limitation of usable eluent and the diffusion of eluted substances in the postcolumn reactor. Thus, there is no satisfactory method for the analysis of triglycerides in terms of resolution, selectivity, sensitivity, and quantitativity. On the other hand, two colorimetric methods, the chromotropic acid method (13) and acetylacetone method ( 1 4 ) , are well-known for the quantitative determination of triglycerides. It is not easy to apply these methods to the postcolumn reaction because of slow reaction rate and high viscosity of a reaction mixture. However, the latter method is superior to the former one in terms of selectivity and sensitivity and can be carried out under mild reaction conditions. By using the latter method, therefore, we tried to develop a
new PCRD with high selectivity, high sensitivity, and molar responsibility for the analysis of triglycerides. This paper describes the optimization of the postcolumn reaction and the analyses of triglycerides of natural fats and oils with high selectivity, high sensitivity, high resolution, and molar responsibility. EXPERIMENTAL SECTION Apparatus. The schematic diagram of the liquid chromatograph with a PCRD is shown in Figure 1. The liquid chromatograph consisted of a reciprocating piston pump (Model 655, Hitachi, Tokyo, Japan), a gradient controller (Model 655-66, Hitachi), a variable-wavelength W-vis detector (UVIDEC 100-111, Japan SpectroscopicCo., Tokyo, Japan), an injector (Model, 7125, Rheodyne, Berkeley, CA),and a circulator (Model FE, Haake Inc., Karlsruhe, West Germany) for column temperature control. The PCRD consisted of three acid-resistant pumps (NP-S-253,Nihon Seimitsu, Tokyo, Japan) and two constant-temperature circulating baths (Model D1-L and D2-L, Haake, Inc.) for hydrolysis and color development reaction. The postcolumn reaction coil was made of Teflon tubing (0.5mm id.). Chromatograms, peak areas, and retention times were obtained by using a data processor (CHROMATOPAC C-RlA, Shimadzu Co., Kyoto, Japan). A UV-vis recording spectrophotometer (Model 340, Hitachi) was used for the optimization of the postcolumn reaction. Reagents. Hitachi gel 3057 was used as a stationary phase, which is ODS packings of average diameter 3 wm. Authentic triglycerides were purchased from Sigma Chemical Co. (St. Louis, MO). Acetonitrile and ethanol of HPLC analysis grade were obtained from Kanto Chemical Co., Inc. (Tokyo, Japan). Other reagents were of analytical reagent grade. In the postcolumn reactor the following reagents were used. For the hydrolysis of triglycerides, 2.4% potassium hydroxide in ethanol-water (60/40) was used. For the oxidation of glycerin, 10 mM periodic acid solution dissolved in ammonium acetate-acetic acid buffer of pH 5.5, which is prepared by mixing 4.0 M ammonium acetate and 1.6 M acetic acid in 1/1ratio, was used. For the color development reaction, 0.2 M acetylacetone solution dissolved in ammonium acetate-acetic acid buffer of pH 5.5 was used. These reagents were renewed every day. Postcolumn Reactor. As shown in Figure 1, potassium hydroxide solution (reservoir 6) is added at a flow rate of 0.4 mL/min to eluate from a chromatographic column, and passed through Teflon tubing (reaction coil 16; 0.5 mm i.d., 20 m long) in a circulating bath (bath 19) kept at 85 "C, where triglycerides are hydrolyzed to form glycerin. Then periodic acid solution (reservoir 7) is added at a flow rate of 0.4 mL/min in order to oxidize glycerin to formaldehyde and passed through Teflon tubing (reaction coil 17; 0.5 mm i.d., 1 m long). Acetylacetone solution (reservoir 8 ) is added t o the flow at a rate of 0.4 mL/min, and the mixture is passed through Teflon tubing (reaction coil 18; 0.5 mm id., 20 m long) in a circulating bath (bath 20) kept at 70 'C to form 3,5-diacetyl-l,4-dihydrolutidine, which is detected at 410 nm with a UV-vis detector. These reactions (14, 15) are carried out continuously within 5 min. Procedure. For isocratic elution, two stainless columns (4.6 mm i.d., 150 mm long) packed with Hitachi gel 3057 were connected together and kept at 35 OC. Ethanol-acetonitrile (60/40) was used as a mobile phase at a flow rate of 0.8 mL/min. For gradient elution, a stainless column (4.0 mm i.d., 250 mm long) was used and kept at 30 "C. The mobile phases used were ethanol-acetonitrile 25/75 (solvent A) and 75/25 (solvent B) at a flow rate of 0.8 mL/min. The analysis was done according to
0 1986 American Chemical Society 0003-2700/86/0358-2380$01.50/0
ANALYTICAL CHEMISTRY, VOL. 58, NO. 12, OCTOBER 1986
Flgwe 1. Schematlc diagram of the liquid chromatograph: 1-4, pump; 5, eluent reservoir; 6, reservoir for potassium hydroxide reagent; 7, reservoir for periodic acid reagent; 8, reservoir for acetylacetone reagent; 9, air damper; 10, pressure gauge; 11, resistant coil (0.25 mm i.d. X 2.5 m); 12, sample injector; 13, analytical column; 14, circulating water bath (30 or 35 "C); 15, three-way mixing joint; 16, reaction coil (0.5 mm i.d. X 20 m); 17, reaction coil (0.5 mm i.d. X 1 m); 18, reaction coil (0.5 mm i.d. X 20 m); 19, water bath (85 "C); 20, water bath (70 "C); 21, UV-vis detector; 22, datalyzer.
0
0
5 HI04
IO concentration (nM)
15
Figure 2. Effect of the concentration of periodic acid on the absorbance at 410 nm: (0) glycerin (15 ppm); (0)reagent blank.
the time program described below. Starting condition is 100/0 (solvent A/B). During 10 min the program rate is kept at 4%/min to 60140 (A/B) and then is changed to 2%/min, which is kept during 30 min to Of100 (A/B). This mobile phase is held for 15 min, and then is changed to the starting condition for reconditioning. The starting condition is held for 15 min. A sample containing 0.2-200 pg of triglycerides dissolved in 5-40 p L of acetone or less than 5 p L of tetrahydrofuran was injected into HPLC. RESULTS AND DISCUSSION Optimization of t h e Separation. The analysis of triglycerides by means of HPLC has been investigated with NARP chromatography using ODS column in almost all cases. Mixtures of acetone, acetonitrile, propionitrile, tetrahydrofuran, chloroform, methylene chloride, methanol, ethanol, and hexane are used as a mobile phase. Among these solvents, mixtures of acetone and acetonitrile are frequently used. In order to investigate NARP chromatography of triglycerides, Hitachi gel 3057 was chosen as a stationary phase because of the high resolution. On the other hand, only a few solvents can be used as a mobile phase because of the interference effects on the postcolumn reaction. Among these solvents, ethanol and acetonitrile were compatible with the mobile phase, and excellent resolution was obtained by using ethanol-acetonitrile (60/40)as a mobile phase. However, long chromatographic columns (250-300 mm long) were used in the present study as described in the Experimental Section, since the chromatographic peaks broadened about two times by the diffusion of eluted substances in the postcolumn reactor. Optimization of Postcolumn Reaction. In order to investigate the postcolumn reaction, each reaction was optimized in the order of oxidation, color development, and hydrolysis
0
0
0.1
0,2
0.3
0+4
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0.5
Acetylocetone tontentratlon ( M I
Figure 3. Effect of the concentration of acetylacetone on the absorbance at 410 nm: (0)glycerin (15 ppm); (0)reagent blank.
t "0
1 I
60
120
\
.
\o a
I
I
I
180
240
300
Reactlon time (sec)
Figure 4. Effect of heating time on the absorbance at 410 nm: (0) glycerin (15 ppm); (0)reagent blank.
reaction. Oxidation and color development reaction were investigated by the manual acetylacetone method (15) with glycerin as a sample instead of triglycerides. Optimization of Oxidation Reaction. Figure 2 shows the effect of the concentration of periodic acid on the absorbance at 410 nm, which is proportional to the concentration of formaldehyde formed, measured by the manual acetylacetone method (15). In this experiment, periodic acid solution was buffered at pH 5.5 with ammonium acetate-acetic acid buffer for color development reaction as described later, though periodic acid oxidation was usually carried out in acetic acid (14). The absorbance increases with an increase in concentration of periodic acid and reached nearly constant value above 10 mM of periodic acid. Thus, 10 mM of periodic acid was used for the oxidation reaction. In addition, this reaction proceeded in a very short time at room temperature, which indicated that only 1 m of Teflon tubing was required. On the basis of these results, periodic acid oxidation was carried out under the conditions described in the Experimental Section. Optimization of Color Development Reaction. Optimum temperature and pH for the color development reaction are 40-60 "C and pH 5.5-6.5, respectively (14, 15). In the present postcolumn reaction, potassium hydroxide is added for the hydrolysis of triglycerides before the periodic acid oxidation. Since lower pH of buffer solution allows higher concentration of potassium hydroxide solution, the pH of the buffer solution was set at 5.5, the lower limit of optimum pH, and the periodic acid solution was also buffered at pH 5.5 in order to increase the buffer capacity. The reaction was carried out at 70 "C, a little higher than the optimum temperature, since high reaction rate was required in the postcolumn re-
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ANALYTICAL CHEMISTRY, VOL. 58, NO. 12, OCTOBER 1986
Table 11. Range of Calibration Curves and Reproducibilities coeff of range of calibration curve, nmol
triglyceride
trilaurin trimyristin tripalmitin tristearin
variation," 9c
0.3-59.9 0.3-50.7 0.5-25.7 1.1-11.2
0.93
1.13 1.80 3.29
Each sample (5.0 fig) is injected and analyzed five times. 0
10
3':
20
Tube length ( m )
Flgwe 5. Effect of the length of Teflon tubing in the postcolumn reactor on peak areas (sample, glycerin).
Table I. Effects of Hydrolysis Conditions on the Responsibility of the Postcolumn Reactor Detector
EtOH/H20 70/30 65/35 70 reaction temp ("C) 70 10 length of tubing (m) 10 re1 molar responsen 1.00 trilaurin 1.00 trimyristin 1.01 0.99 tripalmitin 1.02 1.01 tristearin 0.93 0.87 triolein
60/40 70
60/40 80
10
20
1.00 0.99 0.99
1.00 0.99 1.03 0.93
0.80
60/40 85 20 1.00 0.99 1.00 0.97 1.00 i
(I
Relative molar response to trilaurin.
0
1
5
IO
15 TIN
actor. Figure 3 shows the effect of the concentration of acetylacetone on the absorbance at 410 nm. Since a maximum was observed in the 0.1-0.2 M region, the concentration of acetylacetone was determined to be 0.2 M. Figure 4 shows the effect of reaction time on the absorbance at 410 nm. The absorbance increases with an increase in reaction time and reaches a nearly constant value in 60-120 s. Prolonged reaction brings a decrease in absorbance because of the decomposition of the yellow product, 3,5-diacetyl-1,4-dihydrolutidine. Finally, the effect of the length of Teflon tubing on the color development reaction (peak areas) under the conditions described above was investigated by using the postcolumn reactor. As shown in Figure 5, peak areas rapidly increase with an increase in length of tubing up to 15 m and reach a nearly constant value in the range 15-30 m. Since longer tubing afforded a broader peak because of the diffusion in tubing, the length was set at 20 m. On the basis of these results, the conditions of the color development reaction were determined as described in the Experimental Section. Optimization of Hydrolysis Condition. In order to determine the optimum concentration of potassium hydroxide, the effect of the concentration on the color development reaction was investigated first. Since no effect was observed up to the concentration of 2.470, the potassium hydroxide concentration was fixed at 2.4%. On the other hand, it is most important to establish molar responsibility of the PCRD. Table I shows the responsibility of this PCRD to trilaurin, trimyristin, tripalmitin, tristearin, and triolein under the various conditions. The relative responses of tristearin to trilaurin are a little lower than those of other triglycerides; however an increase in ethanol content in the hydrolysis solution increases the relative molar response of tristearin to trilaurin. Since a few troubles such as ghost peaks and plugging of tubing because of the formation of precipitates were observed above 65% ethanol content, the ethanol content was determined to be 60%. The increases in temperature and reaction time (the length of tubing) also afforded an increase in relative molar response of tristearin to trilaurin, which was 0.97 at 85 "C and 20 m of tubing. On
0 (min)
10
20
30
40
Flgure 6. Chromatograms of a standard mixture of triglycerides and waxes with a UV detector (210 nm) (A) and with the postcolumn reactor detector (B): 1, impurity from trilinolein; 2, trilaurin; 3, CZ8wax; 4, triliilein: 5, C30 wax; 6, trimyristin; 7, C32wax: 8, C, wax; 9, triolein; 10, tripalmitin.
the basis of these results, the hydrolysis condition was determined as described in the Experimental Section, and under these conditions the present PCRD shown in Figure 1indicated molar responsibility to all triglycerides up to tristearin. Calibration Curves and Reproducibilities. The linear range of calibration curves of trilaurin, trimyrisitn, tripalmitin, and tristearin under the optimum conditions is shown in Table 11, and their detection limits are 0.1-0.5 nmol. Reproducibilities of the analyses of 5 pg of each triglyceride were within 3.3% relative. On the other hand, injection solvents severely affect the resolution. Tsimidou et al. (16, 17) recommended acetone as an injection solvent and also described that a large amount (above 20 pL) of the injection solvents such as chloroform and THF decreased the resolution. In the present study, acetone was used as an injection solvent according to Tsimidou et al.; however less than 5 p L of THF was used for the analysis of higher triglycerides such as tristearin because of the poor solubility to acetone. In addition, THF affords a small ghost peak at V,,as shown in Figure 8.
ANALYTICAL CHEMISTRY, VOL.
3
13
20
43
30
50
60
-lme ( m i n ~
Figure 8. Typical chromatogram of a standard mixture of triglycerides with gradiint elution by using the postcolumn reactor detector: 1, peak from THF; 2, tricaprylin; 3, tricaprin; 4, trilaurin; 5, triiyristin; 6, triolein; 7, tripalmitin; 8. tristearin. 56 7
58, NO. 12, OCTOBER 1986
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sponsibility, while a UV detector detected both triglycerides and waxes with higher response to unsaturated compounds. An example of the analysis of triglycerides of natural fats and oils by the present method is shown in Figure 7. Gradient Elution. The present PCRD permits the use of gradient elution without drift of base line and with molar responsibility as shown in Table 111. Figure 8 shows the analysis of a standard mixture of triglycerides, from tricaplyrin to tristearin. The better and satisfactory resolution of triglycerides of coconut oil was obtained as shown in Figure 9, compared with that of isocratic elution in Figure 7 . Therefore, the present PCRD system should offer a versatile method for the analysis of mixtures of triglycerides. ACKNOWLEDGMENT
The authors wish to acknowledge the technical assistance of Hideko Ono and Kunio Iida. Registry No. Trilaurin, 538-24-9; trimyristin, 555-45-3;tripalmitin, 555-44-2; tristearin, 555-43-1; triolein, 122-32-7; tricaprylin, 538-23-8; tricaprin, 621-71-6; 3,5-diacetyl-1,4-dihydrolulidine, 1079-95-4. LITERATURE CITED
Table 111. Responsibility of the Postcolumn Reactor Detector for the Gradient Analysis
triglyceride
re1 molar response to trilaurin
tricaprylin tricaprin trilaurin trimyristin tripalmitin tristearin
0.98
triolein
1.01 1.00
1.03 1.01 0.99 1.03
Application. Figure 6 shows the analysis of a mixture of triglycerides and waxes and also shows the selectivity of the present PCRD comparing with a UV detector (210 nm). The present PCRD detected only triglycerides with molar re-
(1) Dong, M. W.; Dicesare, J. L. J. Am. Oil Chem. SOC. 1983, 6 0 , 788. (2) Phillips, F. C.; Erdahl, W. L.; Nadenicek, J. D.; Nutter, L. J.; Schmit, J. A.; Privett, 0. S. Lipids 1984, 19. 142. (3) Phillips, F. C.; Erdahl. W. L.; Schmit, J. A,; Privett. 0. S. Lipids 1984, 19, 880. (4) Parris, N. A. J. Chromatogr. Sci. 1979, 17, 541. (5) Robinson, J. L: Macrae, R. J. Chromatogr. 1984, 303, 386. (6) Robinson, J. L.; Tsimidou, M.; Macrae, R. J. Chromatogr. 1984, 324, 35. (7) Stolyhwo, A.; Colin, H.; Guiochon, G. Anal. Chem. 1985, 57, 1342. (8) Plattner, R. D.; Spencer, G. F.; Kieiman, R. J . Am. Oil Chem. SOC. 1977, 54,511. (9) El-Hamdy, A. H.; Perkins, E. G. J. Am. Oil Chem. SOC. 1981, 58, 867. (10) Singleton, J. A.; Pattee, H. W. J. Am. OiiChem. SOC. 1984, 67.761. (11) Compton, B. J.; Purdy, W. C. Anal. Chim. Acta 1982, 742,13. (12) Compton, B. J.; Purdy, W. C. Anal. Chim. Acta 1980, 119, 349. (13) Handel, E. V.; Ziiversmit, D. B. J. Lab. Ciin. Med. 1957, 5 0 , 152. (14) Fletcher, M. J. Clin. Chim. Acta 1968, 22,393. (15) Nash, T. Biochem. J . 1953, 55,416. (16) Tsidmidou, M.; Macrae, R. J. Chromatogr. 1984, 285, 178. (17) Tsimidou, M.; Macrae. R. J. Chromatogr. Sci. 1985, 2 3 , 155.
RECEIVED for review April 15, 1986. Accepted June 6, 1986.
