ANALYTICAL CHEMISTRY, VOL. 50, NO. 2, FEBRUARY 1978
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intensities to the background was l : l 5 . Furthermore, suspended particles occasionally caused "spikes" in the spectrum as they moved through the laser beam. However, a spectrum with a signal-to-noise ratio of 10 was obtained a t a concentration of 444 ppb. Using the criterion of having three bands with a signal-to-noise ratio of >3, we can identify the dye in the seawater and the river water a t the 200-ppb level. T h e results of this study show that Raman spectrometry is a viable method for detection and identification of chemicals in water when those chemicals exhibit resonance Raman scattering. The increased scattering caused by the resonance effect makes it possible to obtain reasonable spectra even when the spectral background is relatively high as in natural water samples.
317
LITERATURE CITED (1) (2) (3) (4)
-
F. B. Bradley and C. A. Frenzel, Waler Res., 4, 1 2 5 (1970). S. F. Baldwin and C. W. Brown, Water Res., 6, 1601 (1972).
M. Ahmadjian and C. W. Brown, Environ. Sci. Techno/.,7 , 452 (1973). K. M. Cunningham, M. C. Goldberg, and E. R. Weiner, Anal. Chem., 49, 70 (1977). ( 5 ) G. Brauniich, G. Gamer, and M. S. Petty, Water Res., 7 , 1643 (1973). (6) C. W. Brown and P. F. Lynch, J , FoodSci., 41, 1231 (1976). (7) L. de Gdan, "Analytical Spectrometry", Adam Hilger Ltd., London, 1971.
RECEIVED for review April 2 7 , 1977. Accepted October 13, 1977. This research was partially supported by the National Sea Grant Program, National Oceanic and Atmospheric Administration. One of us (L.V.H.) gratefully acknowledges financial support from the State University Center of Antwerp and a N.A.T.O. Research Fellowship.
Analytical Chemistry of Amygdalin Thomas Cairns," Jerry E. Froberg, Steve Gonzales, William S. Langham, and John J. Stamp U S . Food and Drug Administration, Los Angeles, California 900 15
John K. Howie and Donald T. Sawyer Department of Chemistry, University of California, Riverside, California 9252 1
High performance liquid chromatography, carbon-13 nuclear magnetic resonance, chemical ionization mass spectrometry, and gas chromatography with flame ionization detection have been employed for the identification and quantitative determlnation of the epimers of amygdalin. The results indicate that a combination of techniques is required to understand total sample profiles and the pharmaceutical composition of current drug samples. Notably, injectable preparations of amygdalin are epimeric and below declared concentration, while tablet dosage forms contain epimerically pure (R)-amygdalin.
History. Amygdalin (I),a naturally occurring cyanogenetic glycoside (1) found in the kernels or seeds of members of the Rosaceae (almond, apple, apricot, cherry, peach, pear, plum, quince), was first isolated in 1830 (2). Subsequently it was discussed that amygdalin could be hydrolyzed by a nitrogenous enzyme associated with the glycoside in the almond. The hydrolysis product, "oil of bitter almonds", was in fact a mixture of benzaldehyde, hydrocyanic acid, gentiobiose, and glucose (3). T h e structural elucidation of amygdalin (I) and various synthetic approaches were first published in 1923/1924 by a number of workers ( 2 , 4 , 5 ) . About ten years later, the biochemistry of amygdalin as well as the physical, chemical, and physiological characterization of the compound and its hydrolysis products, notably HCN, was discussed by Viehoever and Mack (3). Stereochemistry. Amygdalin (I), a gentiobioside of mandelonitrile, contains several chiral (asymmetric) centers which potentially can give rise to a large number of epimeric species. The chiral centers of the gentiobiose moiety, however, are well defined (derived from P-D-glucose) and ai-e stable. The aglycone entity or nonsugar derived chiral center of mandelonitrile is susceptible to epimerization, particularly under basic conditions, because of the weakly acidic character of the benzylic proton. Because these epimers (derivatives of ( R ) 0003-2700/76/0350-0317$01 .OO/O
and (SI-mandelonitrile) will have different physiochemical properties and possible different pharmacological and toxicological properties, analytical procedures for amygdalin should be capable of their identification and quantitative determination. The naturally occurring amygdalin that is extracted from the kernels and seeds of members of the Rosaceae has the R configuration. Laetrile vs. Amygdalin. To avoid any risk of the further proliferation of confusion in nomenclature, amygdalin is not structurally synonymous with Laetrile. Amygdalin (I)
(I) Amygdalin cZOH27N011 0-nrndclonitrile- b e t a - O - g l r c o r l d o - 6 - b ~ t ~ - D - g l ~ c o r l d ~
,
COOH
(11)
Laetrile c14H151107 I-mandolonitrile-beta-glucuronic
acid
is a naturally occurring cyanogenetic glycoside that is present in the kernels of almonds and related fruits. Laetrile (111, according to the patents issued (6, 7 ) results (a) from hydrolysis of amygdalin and subsequent oxidation of the Lmandelonitrile-&glucoside product with platinum black, or C 1978 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 50, NO. 2, FEBRUARY 1978
Table I. Analysis of AmygdalinGontaining Samplesa
No.
1 2a 2b 3
4a 4b 5a 5b 5c a
Tablet Tablet Tablet Injectable preparation Injectable preparation
Found, % Wt, g
Declared
R
S
15
3 g/lO m L
72 70 32 26
0 0 0 0 38
0.70
3 g/10 mL 3 g/10 mL
28 12
38 16
0.73 0.75
3 g/10 m L 3 g/10 mL
19 8
28 10
0.86 0.64 0.65 0.41
Analysis made on different lots of same product.
RIS
GCIFID, Found, % S RIS
HPLC, Found, %
I3C-NMRb
Sample type
R
S
RIS
R
14 68
0 0
100
25
35
0.72
72 24 41
0 0 0 0 54
24
35 19
0.70
32
43
0.74
11
0.58
9
21
0.43
0.70
18
27
0.76
7
11
0.67 0.62
26 14
33 18
0.78 0.77
27
0.75
Based solely on purity of organic matter assay.
(b) by condensation of mandelonitrile with glucose followed by oxidation, or (c) by condensation of mandelonitrile with glucuronic acid. All attempts to prepare Laetrile (11) by the patented procedures have been unsuccessful (8). Recently, however, the synthesis of Laetrile in a one-step procedure by use of a solid phase immobilized glucuronyltransferase has been accomplished (9). The failure of the patented synthetic procedures as well as the great expense required to achieve the enzymatic synthesis of Laetrile established that this trade-marked compound exists only in extremely limited quantities. The proponents of Laetrile and the popular press have confused the situation by their use of this name to describe amygdalin (I). Presently, the drug that is offered as a n anticancer agent is amygdalin (I). In this paper, we report analytical protocols for the specific identification and quantitation of amygdalin and its epimers by use of HPLC, I3C-FTNMR, GC/FID, and GCMS. EXPERIMENTAL Reagents. (a) Acetonitrile. Nanograde. (b) Ethanol. 95% and absolute. (c) Trifluoroacetic Acid Sodium Salt. Eastman Organic Chemicals, Rochester, N.Y. (d) Trifluoroacetic Anhydride. Available from Eastman Organic Chemicals, Rochester, N.Y. (e) Reaction Mixture. Trifluoroacetic anhydride was mixed with an equal volume of acetonitrile that contained 10% trifluoroacetic acid, sodium salt. (f) Petroleum Ether. Pesticide grade. (g) Amygdalin. Aldrich Chemical Co. The material was recrystallized by dissolving 2 g in 100 mL 95% ethanol that was heated to boiling. The resulting solution was filtered twice through a fritted glass filter funnel, prior to its evaporation to 20 mL, and allowed to recrystallize. The resulting trihydrate crystals were then dissolved in 100 mL of boiling absolute ethanol and concentrated to less than 10 mL. After recrystallization, the crystals were dried in a 60 "C vacuum oven for 18 h (mp 215 "C) and stored in a desiccator. Addition of 1 drop of aqueous ammonia (5 mL concentrated ammonia diluted to 25 mL) to an amygdalin sample solution (0.4 8/51 mL) resulted in an epimeric mixture. Apparatus. (a) Gas Chromatograph. Equipped with a flame ionization detector with (1)a 6 f t X 3.5 mm i.d. glass column packed with 2% OV-101 on CWHP; operating conditions: purified nitrogen flow 60 mL/min; temperatures, column 220 "C, detector 250 "C, injection 220 "C; (2) a 6 f t X 2 mm i.d. glass column packed with 2% F-50 versilube on CWHP; operating conditions: purified nitrogen flow 30 mL/min; temperatures, column 180 "C, detector 300 "C, injector 250 "C. (b) Gas Chromatograph-Mass Spectrometer. Finnigan Model 3300 with Data System 6000 or equivalent. Operating conditions: 5 f t X 2 mm i.d. glass column packed with 3% OV-101 on Chromosorb 750,100-120 mesh, methane as carrier and reagent gas at 30 mL/min; temperatures, column 220 "C, source 150 "C, injector 250 "C; MS accelerating voltage, 150 V; MS inlet pressure, 1 Torr. (c) '3C-Nuclear Magnetic Resonance Spectrometer. %-NMR spectra were recorded on a Bruker WH-90 pulsed Fourier
Transform multinuclear NMR spectrometer or equivalent equipped with quadrature phase detection. The spectrometer was internally locked on the deuterium resonance of the solvent, 1:4 ( v i v ) D,O:H,O. The fixed frequency transmitter (22.638 MHz), probe head, and preamp were used. Two 8K free induction decays (FIDS) were collected at a sweep width of 5000 Hz, yielding an acquisition time of 1.638 s and digital resolution of 0.61 Hz. Satisfactory signal to noise ratio was obtained after 2200 4.5-ps pulses (90" pulse, 13.4 p s ) ; total data collection time, 1 h. Exponential multiplication was performed on the FIDS prior to transformation by use of a line broadening constant of 0.5 Hz. (d) Liquid Chromatograph. A Spectro-Physics Model 3500 or equivalent equipped with 250 X 3 mm i.d. 10-pm spherisorb ODS column and UV detector at 254 nm was used at ambient temperature. Operating conditions: (a) for the quantitative determination of the R form and to screen samples for epimers, solvent 4% CH3CN/H20, flow rate 1.6 mL/min; (b) for the quantitative determination of R and S forms, solvent 0.2% CH3CNjH20,flow rate 1.2 mL/min. Sample Preparation. (a) Injectable Preparations. A quantity of sample equivalent to 10-30 mg amygdalin was diluted with distilled water for HPLC determination or derivatized for GC determination (10). (b) Tablets and Powders. A sample equivalent to 10-30 mg of Amygdalin was weighed and diluted with distilled water prior to filtration through 0.45-bm Millipore filter for HPLC determination. For GC analysis, the diluted sample was extracted according to the procedure in the following paragraph. (c) Fatty Products. Samples were ground to pass through a 30-mesh sieve, then weighed and extracted twice with 100 mL of petroleum ether. Sufficient sample was used to yield 10-30 mg of amygdalin. The solid portion was filtered with a glass fritted funnel and then extracted twice with 60-mL portions of boiling 95% ethanol, boiling gently for 2-3 min each time. The resulting solution was filtered through a glass fritted funnel and then evaporated to near dryness with the aid of vacuum and a heat source. With the aid of two portions of absolute ethanol, the product was recrystallized. The concentrations were adjusted to allow amounts injected within the linear range between 400 to 2000 ng for both HPLC and GC. RESULTS A representative sampling of amygdalin tablets and injectables that currently are manufactured in Mexico has been examined by a combination of techniques-HPLC, GC/FID, GCMS, and 13C-NMR (Table I). Gas Chromatography. Because amygdalin has gentiobiose as the parent glycoside, the present investigation has utilized the technique of trifluoroacetic acid derivatization (TFA) for GC analysis as previously suggested for mono- and disaccharides in cocoa products (IO). Methodology has been developed for the extraction and clean-up of amygdalin from food products, tablets, and injectables. The increased volatility of TFA-amygdalin relative to the corresponding TMS derivative is noteworthy. On a nonpolar column such as OV-101
ANALYTICAL CHEMISTRY, VOL. 50, NO. 2, FEBRUARY 1978
319
I"
I
a
I 1
a
J
b 8-Amygdalin
\
\
b
C
Arnypdalln
Time in mina
n
I,
-D
Flgure 1. GC separation of TFA derivatives of (R)and (S)-amygdalin: (a) tablet, (b) injectable preparation. GC conditions: 6 ft X 2 mm i.d. 2 % versilube F-50 column, 30 mL/min Npr 180 O C isothermal
TFA-amygdalin chromatographs in about 3.75 min at 220 "C as compared to 5 min at 280 "C for the TMS-amygdalin (11). However, separation of the epimers of amygdalin could not be achieved on OV-101 by use of TFA derivatives. Nahrstedt (11) has reported a partial separation of (R)- and (S)-amygdalin (as their TMS derivatives) on both OV-1 and OV-17. In an attempt to resolve the epimers as their TFA derivatives, conditions have been optimized for the OV-210 stationary phase. A partial separation has been achieved, but the experimental conditions caused both column bleed and decomposition of the TFA derivatives. Subsequently, a 2% F-50 (G.E. versilube, 10% trichloropheny1:methyl silicone) column has proved to be effective for the separation of the amygdalin epimers as their TFA derivatives (Figure 1). The McReynold's constant (12) for versilube F-50 is about the same as OV-101 (229 and 240 respectively) while that of OV-210 is 1520. Hence, the effectiveness of F-50 for the epimeric separation is surprising in view of the failure of OV-101 to resolve the peaks. The success of F-50 must be due to a specific interaction of the TFA groups with the trichlorophenyl entity of the stationary phase. G a s Chromatography-Mass Spectrometry. In an attempt to establish the identity of the TFA derivative of amygdalin, GC-MS has been employed. Figure 2 illustrates the separation of the mono- and disaccharides (apparently present in original samples) from amygdalin in various products. The elution time of TFA-amygdalin relative to the other sugars indicates that amygdalin can be derivatized intact and chromatographed as such. The observance of the parent ion for derivatized amygdalin (1129 amu) with chemical ionization has not been possible because the mass spectrometer system has a limit of 1000 amu. However, the presence of abundant ions a t m l e 319, 547, 769, and 883 indicates that the derivatized amygdalin is an intact species. The gas chromatographic and GC-MS studies provide convincing
d 0 100
5
10
15 mins
150
200
250oc
Figure 2. GCMS Total Ion Chromatograms of TFAderivatized extracts o
