Anal. Chem. 1998, 70, 1437-1439
Technical Notes
Selective HPLC Analysis of n-Alkyl Hydroperoxides up to C18H38O2 Petra Heinmo 1 ller,† Hans-Hagen Kurth,‡ Richard Rabong,§ Walter V. Turner,⊥ Antonius Kettrup,† and Siegmar Ga 1 b*,⊥
GSFsInstitut fu¨ r O ¨ kologische Chemie, Schulstrasse 10, 85356 Freising-Attaching, Germany, FraunhofersInstitut fu¨ r Umweltchemie und O ¨ kotoxikologie, Auf dem Aberg 1, 57392 Schmallenberg, Germany, GSFsInstitut fu¨ r Biochemische Pflanzenpathologie, Ingolsta¨ dter Landstrasse 1, 85764 Oberschleissheim, Germany, and Analytische Chemie, Bergische Universita¨ t, Wuppertal, 42119 Wuppertal, Germany
A series of n-alkyl hydroperoxides are separated by HPLC and detected by their postcolumn reaction with horseradish peroxidase and p-hydroxyphenylacetic acid (HPAA) to yield a fluorescent product; several secondary and tertiary hydroperoxides, some 1-hydroxyalkyl hydroperoxides, and a few branched hydroperoxides are also examined. n-Alkyl hydroperoxides up to at least C-18 react with the enzyme with only minimally reduced efficiency at greater alkyl chain length. The effects of the column, the eluent, and the pH of the sample reaching the detector are described. The detection limit with gradient elution ranges from 0.4 µmol L-1 for n-hexyl hydroperoxide to 1 µmol L-1 for n-octadecyl hydroperoxide. Reported methods for HPLC analysis of hydroperoxides differ essentially in the detection system, postcolumn reactions having been chosen by most groups analyzing environmental and biological samples on account of their sensitivity and selectivity. One of the systems in most common use is the chemiluminescence resulting from the reaction of peroxides with the combination of microperoxidase-11 (MP-11) and either luminol or isoluminol, for detection of primary, secondary, and tertiary alkyl hydroperoxides with a detection limit of a few picomoles.1 Unfortunately, MP-11 is expensive and not very selective, a number of nonperoxidic species (for example, the ubiquinols) giving interferences;1 antioxidants are reported to give negative peaks in HPLC analyses with this method.2 The combination of horseradish peroxidase (HRP) and phydroxyphenylacetic acid (HPAA) with fluorescence detection has been used to determine both H2O2 and hydrophilic alkyl hydroperoxides.3-7 While this method requires a rather complex GSFsInstitut fu ¨r O ¨ kologische Chemie. FraunhofersInstitut fu ¨ r Umweltchemie und O ¨ kotoxikologie. § GSFsInstitut fu ¨ r Biochemische Pflanzenpathologie. ⊥ Bergische Universita ¨t. (1) Yamamoto, Y.; Frei, B.; Ames, B. N. Methods Enzymol. 1990, 186, 371380. (2) Yamamoto, Y.; Ames, B. N. Free Radical Biol. Med. 1987, 3, 359-361. (3) Hellpointner, E.; Ga¨b, S. Nature 1989, 337, 631-634. † ‡
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© 1998 American Chemical Society
instrumental setup, its advantages are likely to outweigh that consideration; there have also been studies of ways of simplifying the instrumentation.8 HRP is almost completely selective for H2O2 and n-alkyl hydroperoxides, although it reacts with a few shortchained, unbranched secondary hydroperoxides.9 The detection limit for H2O2 in HPLC systems based on the HRP/HPAA reaction is 5 × 10-8 mol L-1, which corresponds to 1 pmol in a 20-µL sample. Of methods selective for hydroperoxides, this is among the lowest yet known. The use of MP-11/HPAA/fluorescence detection10-12 broadens the range of hydroperoxides that can be detected, but, as mentioned above, cost and selectivity become important when MP-11 is used. Despite the active interest in this area, there are no studies in the literature of the application of any of these methods to the analysis of a homologous series of organic hydroperoxides. We report here the use of HPLC with HRP/HPAA detection to determine n-alkyl hydroperoxides from C-4 to C-18 as well as 1-hydroxyalkyl hydroperoxides and several secondary hydroperoxides. The system is optimized for the separation, the postcolumn reaction, and the fluorescence detection. We also discuss the effect of the structure of hydroperoxides on the efficiency of the detection system. EXPERIMENTAL SECTION Chemicals. HPAA, HRP, and the chemicals used as buffers, as solvents, or in syntheses were supplied by Merck (Darmstadt, (4) Gunz, D. W.; Hoffmann, M. R. Atmos. Environ. 1990, 24A, 1601-1633. (5) Lee, J. H.; Leahy, D. F.; Tang, I. N.; Newman, L. J. Geophys. Res. 1993, 98 (D2), 2911-2915. (6) Ga¨b, S.; Turner, W. V.; Kurth, H.-H. In Atmospheric Oxidation Processes; Becker, K. H., Ed.; EC-Air Pollution Research Report 33; E. Guyot SA: Brussels, 1990; pp 41-44. (7) Hewitt, C. N.; Kok, G. L.; Fall, R. Nature 1990, 334, 56-58. (8) Kurth, H.-H.; Ga¨b, S.; Turner, W. V.; Kettrup, A. Anal. Chem. 1991, 63, 2586-2589. (9) Paul, K. G.; Ohlsson, P. I.; Wold, S. Acta Chem. Scand. B 1979, 33, 747754. (10) Ba¨chmann, K.; Hauptmann, J.; Polzer, J.; Schu ¨ tz, P. Fresenius J. Anal. Chem. 1992, 342, 809-812. (11) Kurth, R. H.-H. Doctoral Dissertation, Technical University of MunichsWeihenstephan, 1992. (12) Rabong, R. Master’s Dissertation, Ludwig Maximilian’s University, Munich, 1991.
Analytical Chemistry, Vol. 70, No. 7, April 1, 1998 1437
Germany). Acetonitrile and methanol were Lichrosolv grade. MP11 was from Sigma (Deisenhofen, Germany). Water for eluent and reagent solutions was deionized and doubly distilled. Eluent and reagent solutions were prepared fresh each day and degassed with helium. The n-alkyl, 2-butyl, 3-pentyl,13 3-cyclohexenyl, 3-cycloheptenyl, and R- and β-pinenyl hydroperoxides14 have all been described previously in the literature. 1-Hydroxyalkyl hydroperoxides were made by the reaction of H2O2 with the corresponding aldehyde;15 they were not purified. 2-Hydroxyethyl hydroperoxide resulted from the TiO2-catalyzed reaction of H2O2 with ethylene oxide, and a mixture of 2-hydroperoxy propanol and 2-hydroxypropyl hydroperoxide was produced when this reaction was carried out on propylene oxide. The method is that of Adam and Rios,16 but the preparation of these three hydroperoxides has not been described; the two hydroxypropyl hydroperoxides were not separated. The purity and structures of the alkyl and β-hydroxyalkyl hydroperoxides were determined by 13C NMR and/or 1H NMR (to be published); the purity was confirmed by iodometric titration. Apparatus. The following columns were used: column 1, RP18, 250 mm × 4.6 mm, ODS-Hypersil, 5-µm beads (Shandon); column 2, RP-18, 125 mm × 4.6 mm, ODS-Hypersil, 5-µm beads (Shandon); column 3, RP-8, 250 mm × 4.6 mm, MOS-Hypersil, 5-µm beads (Shandon); and column 4, RP-8, 125 mm × 4 mm, RP select-B, 5-µm beads (Merck). The instrumental setup and the HPLC hardware for isocratic elution were as described in an earlier report.3 The analyses with gradient elution were carried out with a somewhat modified system, in which the first reagent pump after the column delivered a solution of HRP and HPAA and the second a solution of NaOH:17 eluent, acetonitrile or methanol in various proportions in dilute H3PO4 (pH 3.5), flow rate 0.5 mL min-1; reagent 1, 20 mg of horseradish peroxidase and 4 mg of p-hydroxyphenylacetic acid in 250 mL of 0.01 M KH2PO4 (pH 7.0), flow rate 0.5 mL min-1; reagent 2, 0.03 M NaOH, flow rate 0.1 mL min-1; injected volume, 20 µL; fluorescence detector, Hewlett-Packard model 1046A or Merck/Hitachi model F-1080, λEx ) 295 nm, λEm ) 415 nm. RESULTS AND DISCUSSION In the reversed-phase HPLC analysis with HRP/HPAA detection, the hydroperoxides are separated as such. The column effluent is then mixed with a solution of HPAA and HRP; in the HRP-catalyzed reaction, hydroperoxides oxidize HPAA to a biphenyl derivative that, after addition of NaOH, is detected by its fluorescence.18 Optimizing the system thus involves optimizing the separation, the postcolumn reaction, and the detection. Optimization of the Separation. Isocratic elution with methanol or acetonitrile in acidified water can be optimized for separation of either the hydroperoxides with shorter alkyl chains or those with longer chains. H2O2, methyl, ethyl, and the two isomeric propyl hydroperoxides,8 the various hydroxypropyl (13) Williams, H. R.; Mosher, H. S. J. Am. Chem. Soc. 1954, 76, 2984-2990. (14) Schenk, G. O.; Eggert, H.; Denk, W. Liebigs Ann. Chem. 1953, 584, 177198. (15) Rieche, A.; Meister, R. Chem. Ber. 1933, 66, 718-727; 1935, 68, 14651473. (16) Adam, W.; Rios, A. J. Chem. Soc., Chem. Commun. 1971, 822. (17) Kok, G. L.; Hewitt, C. N. J. Atmos. Chem. 1991, 12, 181-194. (18) Guilbault, G. G.; Brignac, P. J., Jr.; Juneau, M. Anal. Chem. 1968, 40, 12561263.
1438 Analytical Chemistry, Vol. 70, No. 7, April 1, 1998
Table 1. Retention Times of Hydroperoxides on Isocratic Elution with 70% (v/v) Acetonitrile in Water at pH 3.5 retention time (min) hydroperoxide
column 1
column 2
column 3
n-butyl n-pentyl n-hexyl n-octyl n-decyl n-dodecyl n-tetradecyl n-hexadecyl
7.2 8.0 9.4 14.9 28.3 61.8
2.6 2.9 3.3 4.7 8.1 15.7
7.8 13.7 34.8 61.1 82.0
hydroperoxides, and 1-hydroxybutyl hydroperoxide are eluted from an octadecylsilane (RP-18) column3 by dilute phosphoric acid (pH 3.5) in less than an hour, but n-butyl hydroperoxide is not. Mixtures of acetonitrile or methanol with dilute phosphoric acid (pH 3.5) were used in attempts to obtain baseline separation of groups of hydroperoxides within a reasonable time. Table 1 shows the retention times of n-alkyl hydroperoxides in the range butyl hydroperoxide to hexadecyl hydroperoxide on isocratic elution with a 70:30 (v/v) mixture of acetonitrile and water at pH 3.5. The separation is good on column 1 (250 mm), but the retention time of dodecyl hydroperoxide is 62 min. Reducing the column length (column 2, 125 mm) reduces the retention times of dodecyl hydroperoxide to 16 min but, unfortunately, results in incomplete separation of the lower alkyl hydroperoxides, C-4 to C-6. The more polar column 3 (250 mm, RP-8) also reduces the retention times, making the analysis of n-hexadecyl hydroperoxide possible in 82 min. With methanol-pH 3.5 water mixtures as eluents, the volume fraction of the organic solvent had to be much higher in order to yield comparably rapid analyses. With 95% methanol, the analysis time for a mixture of C-12 to C-18 hydroperoxides was 49 min on column 1. This time could, however, be shortened to 21 min by the use of the more polar column 3. Since no isocratic system capable of eluting octadecyl hydroperoxide (ODHP) from an HPLC column can be expected to separate hydroperoxides with as few as six carbon atoms, we examined gradient elution. The elution of the hydroperoxides from the column and the postcolumn derivatization reactions are coupled systems, since the high percentages of organic solvent necessary to elute the more lipophilic compounds can either reduce the reactivity of the enzyme or lower the efficiency of the fluorescence. Methanol was found to have a less deleterious effect on the sensitivity of detection than acetonitrile. Table 2 shows the retention times of alkyl hydroperoxides on columns 1, 3, and 4 with methanol and acetonitrile gradients. Excellent results were given by a gradient of 80% methanol in dilute phosphoric acid at pH 3.5, rising over the first 6.5 min to a constant 95% methanol; this separates all the n-alkyl hydroperoxides with even-numbered carbon chains from C6H14O2 to ODHP in 18 min on column 4 (Figure 1). An acetonitrile gradient (65% rising to 80% over five minutes) effects the same separation on this column, but the elution of ODHP requires 30 min; this fraction of acetonitrile rather drastically reduces the sensitivity
Table 2. Retention Times of Hydroperoxides on Gradient Elution retention time (min)a hydroperoxide n-hexyl n-octyl n-decyl n-dodecyl n-tetradecyl n-hexadecyl n-octadecyl
column 3 column 4 column 1, CH3OHb CH3OHc CH3CNd CH3OHc CH3CNe 7.7 9.7 13.9 18.5 23.1 28.7 36.3
7.6 9.2 11.8 15.4 18.5 21.2 24.1
5.6 8.4 12.6 18.0 26.3 40.2 64.5
4.1 5.3 7.6 11.1 13.9 15.9 17.6
4.9 7.0 10.1 13.9 18.6 25.7 36.9
a Shown for each column is the organic component of the eluent. From 0 to 6.5 min, linear increase from 85 to 95% methanol, thereafter isocratic. c From 0 to 6.5 min, linear increase from 80 to 95% methanol, thereafter isocratic. d From 0 to 5 min, linear increase from 60 to 70% acetonitrile, thereafter isocratic. e From 0 to 5 min, linear increase from 65 to 80% acetonitrile, thereafter isocratic.
b
Figure 1. Gradient elution of n-alkyl hydroperoxides from column 4 (RP-8, 125 mm × 4 mm, RP select-B, 5-µm beads). Eluent: 80% methanol in dilute phosphoric acid at pH 3.5, rising over the first 6.5 min to a constant 95% methanol.
of the detection, and progressive loss of sensitivity makes higher acetonitrile concentrations impractical. The separation of the same series of hydroperoxides on column 3 is similar to that on column 4 when the 80% to 95% methanol gradient is used, but again the retention time of ODHP is longer (24 min). With this gradient on column 1, this time is extended to 36 min. The detection limits of the hydroperoxides C-6 to C-18, as analyzed by elution with gradients of methanol and acetonitrile, were determined from calibration curves in accordance with German Industrial Standard (DIN) 32645.19 The detection limits of the C-6 to C-16 hydroperoxides with the methanol gradient (column 4) are similar and, on the average, 0.4 µmol/L; for ODHP this limit was higher, at 1 µmol/L. The sensitivity is significantly lower with the acetonitrile gradient: the average of the detection limits of all the n-alkyl hydroperoxides is 3 µmol/L. Optimization of the Postcolumn pH. The intensity of the fluorescence in the fully aqueous HRP/HPAA system is known (19) Deutsche Industrienorm (DIN) 32645, 1994, Deutsches Institut fu ¨ r Normung, Berlin.
to be greater at higher pH.8,20 By examining the detector response to dodecyl, tetradecyl, and hexadecyl hydroperoxides on isocratic elution, we confirmed that this holds when there is 85% methanol in the eluent as well; increasing the pH of the solution reaching the detector above pH 10 brought no significant improvement. Limits on the Range of Hydroperoxides Analyzable with HRP. It is clear from the discussion above that n-alkyl hydroperoxides up to at least ODHP react with HRP. As we reported earlier, 1-hydroxyalkyl hydroperoxides can also be analyzed with this system; these compounds are stable in the acidic eluent, but they are rapidly converted to H2O2 and the corresponding aldehydes when the eluent is made basic after the column,3 and the H2O2 is then detected. The elution times of these 1-hydroxyalkyl hydroperoxides with carbon number Cn are similar to those of the n-alkyl hydroperoxides with Cn-1. Several secondary hydroperoxides were also examined. 2-Propyl hydroperoxide, the lowest secondary hydroperoxide, is detected with sensitivity equal to that of the 1-propyl isomer; the retention times of the two on column 1 with water at pH 3.5 as eluent are 36 and 42 min, respectively.8 The detection limits for 2-butyl and 3-pentyl hydroperoxides with 70% acetonitrile in water at pH 3.5 as eluent were 10 and 12 µmol/L, respectively; in these two cases, the detection limits were ∼50% higher in a system where MP-11 rather than HRP was used with HPAA for detection. We have not had access to higher analogues of these secondary alkyl hydroperoxides, but we have found secondary hydroperoxide isomers of fatty acids and their derivatives not to be detectable with an HRPbased system. It remains to be determined at what chain length the reaction stops or slows down so much that it is no longer useful for detection. 2-Hydroxyethyl, 2-hydroxypropyl, and 1-hydroxy-2-propyl hydroperoxides react rapidly with HRP. Several branched, cyclic, secondary, and tertiary hydroperoxides were examined. 3-Methyl-1-butyl, 3-methyl-2-butyl, cyclohexyl, 3-cyclohexenyl, 3-cycloheptenyl, R-pinenyl, β-pinenyl, tertbutyl, and cumyl hydroperoxides gave not the slightest response with the HRP system, even at concentrations up to 10 mmol L-1. It is, however, possible to analyze all these hydroperoxides if HRP is replaced with MP-11: one has only to sacrifice the sensitivity that HRP gives with the simpler hydroperoxides. We found that substitution of MP-11 for HRP in the fluorescence system (isocratic elution with 70% acetonitrile in water at pH 3.5) gave responses for these branched secondary and tertiary hydroperoxides that were about 1-10% as great as those observed for the n-alkyl hydroperoxides detected with HRP. ACKNOWLEDGMENT Parts of this work were taken from the master’s dissertation of R.R.12 and the doctoral dissertations of H.-H.K.11 and P.H. (Technical University, MunichsWeihenstephan, 1997). We thank Peroxid-Chemie, Ho¨llriegelskreuth, for samples of tert-butyl and cumyl hydroperoxides. Received for review August 13, 1997. Accepted January 16, 1998. AC970867O (20) Lazrus, A. L.; Kok, G. L.; Gitlin, S. N.; Lind, J. A.; McLaren, S. E. Anal. Chem. 1985, 57, 917-922.
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