Spectrophotometric Determination of Pyrrolizidine Alkaloids-Some Improvements SIR: Recently a method was described for the spectrophotometric determination of unsaturated pyrrolizidine alkaloids ( I ) . Some improvements to the original procedure are now described. 1. Because of an error, the solvent for the modified Ehrlich reagent as originally described ( I ) was given as methanol. The results reported in that paper were, in fact, obtained using a reagent made up with absolute ethanol, the only methanol being that which was added with the boron trifluoride. At first, this was not regarded as important, but tests have since shown that the reagent made using methanol gives significantly lower absorbances than one made with ethanol. A further improvement has now been obtained, by replacing the methanolic boron trifluoride with boron trifluoride diethyl etherate, and the revised reagent is described below. 2. The ease with which peroxides formed in the diglyme was a disadvantage of the original method. This has now been eliminated by adding an anti-oxidant, 2,6-di-fert-butyl4-methylphenol (butylated hydroxytoluene; BHT), to the diglyme. Thus protected, the diglyme, if initially free of peroxides, can be kept for long periods without change and, furthermore, it is not affected by hydrogen peroxide carried over from the earlier oxidation stage. 3. Whereas most alkaloids gave excellent reproducible calibration curves up to 100 pg or more, senecionine often gave variable results, especially for the larger amounts. This was traced to the oxidation stage, since the variations were not given by senecionine N-oxide, when the oxidation stage was omitted. The results were improved by certain additions to the oxidation reagent, including small amounts of water, hydroxyl compounds such as glycerol and glycols, and even small pieces of filter paper. These had in common the property of retarding the evaporation of the hydrogen peroxide, so the variable results with senecionine were attributed to incomplete oxidation of the alkaloid. An improved oxidation reagent, containing ethylene glycol and a larger amount of hydrogen peroxide, eliminated the variations with senecionine. Protection of the diglyme in the succeeding stage with BHT, removed the need to eliminate all traces of hydrogen peroxide. The inclusion of BHT in the oxidation reagent also, gave an additional slight improvement. Bingley ( 2 ) found that lasiocarpine also gave erratic results, and obtained a considerable improvement by conducting the oxidation at 77 "C. He regarded thermal decomposition of the alkaloid or N-oxide as a major factor causing the variations. From evidence given below, it appears that both thermal decomposition and incomplete oxidation can contribute to erratic results during the estimation of some alkaloids. EXPERIMENTAL
Absorbances were measured using a Unicam model SP 500 spectrophotometer.
(1) A. R. Mattocks, ANAL.CHEM., 39,443 (1967). (2) J. B. Bingley, ibid., 40, 1166(1968).
Reagents. The oxidation reagent previously described ( I ) will be referred to as oxidation reagent I. Oxidation Reagent 11. In methanol (25 ml) is dissolved 0.25 ml of 30z aqueous hydrogen peroxide (analytical grade, containing 5 mg of sodium pyrophosphate per ml), ethylene glycol (0.25 ml), and BHT (25 mg). For the procedure using isoamyl acetate in place of diglyme, the BHT is omitted. Diglyme is tested for peroxides and if necessary purified as previously described ( I ) . In it is then dissolved BHT, 1 mg per ml. Thus protected, it may be stored for many weeks in a stoppered bottle without further peroxide formation. Modified Ehrlich Reagent. Two milliliters of boron trifluoride diethyl etherate are diluted to 100 ml with absolute ethanol, and in the mixture is dissolved 2 grams of 4-dimethylaminobenzaldeh yde. The Procedure is exactly as previously described ( I ) , but using the above improved reagents. RESULTS
Table I shows the results obtained by plotting calibration curves for a number of alkaloids. The alkaloids were dispensed from methanol solutions, and determinations were usually made in triplicate. The curves were reproducible, and no fall-off or excessive scattering was observed at the higher values, even with senecionine and lasiocarpine. The modified reagents have been in routine use for over a year for determining these and other alkaloids in chemical and metabolic experiments. Use of Isoamyl Acetate in Place of Diglyme. Bingley ( 2 ) found that isoamyl acetate can replace diglyme in the estimation procedure. It is cheaper, and is not subject to oxidation. To evaluate its usefulness in the modified procedure described here, 50-pg lots of four alkaloids were estimated using either diglyme (with BHT) or isoamyl acetate (analytical grade). The results are given in Table 11. In the runs where isoamyl acetate replaced diglyme, BHT was not used, either in the oxidation reagent or in the solvent. Diglyme gave both stronger colors and better reproducibility ; but because of its cheapness and convenience, isoamyl acetate should be useful for routine determinations where the highest sensitivity is not needed. Table I1 also shows the slight improvement which is obtained for retrorsine and senecionine by including BHT in the oxidation reagent. However, only in the results for retrorsine is the improvement statistically significant.
Table I. Calibration Data for Some Pyrrolizidine Alkaloids, from Curves Obtained Using the Improved Reagents Alkaloid
Range, p g
Absorbance for 1 pga
Molar absorptivity
Retrorsine 10-70 0.062 Senecionine 5-100 0.062 Monocrotaline 5-1 15 0.067 Laisocarpine 10-100 0.051 Heliotrine 1c-100 0.063 Rosmarinine 10-100 0.013 For 4-ml sample in I-cm cuvette, at 563 mp rosmarinine).
87,000 83,000
87,000 83,700 79,000 18,400 (562 mp for
VOL. 40, NO. 1 1 , SEPTEMBER 1968
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Table 11. Comparison of Procedures for Estimating Alkaloids Mean Oxidant absorbance, Alkaloido f BHT Solvent* 15 samples f Std dev 0.10 D 3.19 Retrorsine 3.06 0.13 Retrorsine D 2.27 0.23 Retrorsine A 3.36 0.10 D Senecionine 3.29 0.12 Senecionine D 2.25 0.42 Senecionine A 0.10 D 3.37 Heliotrine 2.44 0.28 Heliotrine A 2.48 0.08 D Lasiocarpine 2.12 0.28 Lasiocarpine A = 50 pg in 0.1 ml of methanol. b D = diglyme with BHT; A = isoamyl acetate.
+ + + +
~~
~
Table 111. Hydrogen Peroxide Remaining in Oxidation Reagent Residues after Heating under Various Conditions
Alkaloid, Reagent
100 pg added
None None None Retrorsine Senecionine Lasiocarpine
I I
I1 I I I
Approximate heating temperature, "C 100 77 100 100
100 100
Percentage of ori@nalHzOz remaining after: (minutes) 5 15 30 6.5
80 54 31 10 5.5
0
20 41 0.5 0 0
0 4
31 0 0 0
alkaloids not so heated. Similarly, heating the N-oxide residues of the alkaloids after the oxidation stage caused reductions to 78-87z of the control values. In this experiment, all oxidations were carried out with reagent I at 77 "C for 30 minutes [Bingley's conditions (2)]. Thermal decomposition would seem to account for these differences. 2. Senecionine (100 pg) was treated with oxidation reagent I for 30 minutes at 100 "C, then estimated. Three samples gave a mean absorbance of 1.37. Three further samples, oxidized the same way, were then re-oxidized with reagent I1 at 100 "C for 30 minutes. Estimations then gave a mean absorbance of 4.48. This clearly shows that reagent I did not completely oxidize this alkaloid. Similarly, lasiocarpine after reagent I gave absorbance 2.27, but after reoxidation with 11, 3.39. 3. The hydrogen peroxide remaining after heating oxidation reagents I and 11, with or without alkaloids, was estimated iodimetrically. The results (Table 111) show that at 100 "C hydrogen peroxide evaporates very quickly from reagent I, even when senecionine or lasiocarpine are present. At 77 "C (Bingley's conditions) the peroxide persists much longer, which could account for the improved results from lasiocarpine at this temperature (2). However, with reagent I1 at 100 "C,adequate hydrogen peroxide remains for the full 30-minute period, ensuring complete oxidation of all alkaloids. With retrorsine in reagent I, the hydrogen peroxide persists for longer than with senecionine or lasiocarpine. This may be the reason why retrorsine and many other alkaloids are adequately oxidized by reagent I. The hydrogen peroxide is presumably retained by hydrogen bonding with the alkaloid. ACKNOWLEDGMENT
Causes of Variable Results When Oxidizing Senecionine or Lasiocarpine with Methanolic Hydrogen Peroxide. As already mentioned, erratic results were often obtained when senecionine was oxidized with reagent I, and Bingley (2) had similar difficulties with lasiocarpine. The following experiments were performed to find the causes of these variations.
1. When test tubes containing 50-pg lots of senecionine or lasiocarpine (dry, evaporated from methanol solutions) were heated for 30 minutes in a boiling water bath, then estimated, the readings were only 71-93z of those given by
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ANALYTICAL CHEMISTRY
The author thanks J. B. Bingley for permission to make use of his results prior to publication, and B. A. J. Alexander for technical assistance. A. R. MATTOCKS Toxicology Research Unit Medical Research Council Laboratories Woodmansterne Road Carshalton, Surrey, England RECEIVED for review April 4, 1968. Accepted May 28, 1968.
