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Purification and Quantitative Analysis of Veratridine and Cevadine

by HPLC
J. Dani el Hare
Department of Entomol ogy, Uni versi ty of Cal i forni a, Ri versi de, Cal i forni a 92521
As part of a study to determi ne the vari ati on i n the effecti veness of commerci al pesti ci de formul ati ons
of ester steroi dal al kal oi ds from sabadi l l a (Schoenocaulon officinaleGrey) seeds i n the i ntegrated
pest management of ci trus, a method to i sol ate and measure l arge quanti ti es of al kal oi ds from natural
starti ng materi al s was needed. The addi ti on of the sol vent modi fi er sodi um dodecyl sul fate (SDS)
to a previ ousl y publ i shed HPLC method greatl y i mproved peak resol uti on by mi ni mi zi ng peak tai l i ng.
The use of the non-ester al kal oi d papaveri ne as an i nternal standard permi tted quanti fi cati on of
sabadi l l a al kal oi ds by HPLC, and thi s modi fi ed method was then used to moni tor the breakdown
of cevadi ne and veratri di ne, the two most abundant sabadi l l a al kal oi ds, i n aqueous sol uti ons as a
functi on of ti me and pH.
Keywords: Veratridine; cevadine; veratrine; HPLC
I NTRODUCTI ON
Steroi dal al kal oi ds of the veratrum group are found
i n pl ants of the fami l y Li l i aceae and have a rel ati vel y
ri ch pharmacol ogi cal hi story. These al kal oi ds have been
used as hypotensi ve agents, al though the natural
products now have been superseded by syntheti c ana-
l ogues. However, veratri di ne, one of the al kal oi ds of
thi s group, sti l l has a rol e i n physi ol ogi cal i nvesti gati ons
on the structure and functi on of i on channel s [e.g., Wang
et al . (1990)]. I n addi ti on to thei r rol e i n medi ci ne, crude
mi xtures of veratrum al kal oi ds from seeds of sabadi l l a,
Schoenocaulon officinale Grey, have been used si nce
prehi stori c ti mes as a natural i nsecti ci de (Crosby, 1971).
Formul ati ons of sabadi l l a seeds once agai n are recei vi ng
consi derabl e attenti on i n pest management because of
thei r l ow toxi ci ty to natural enemi es (Bel l ows et al .,
1985; Morse and Bel l ows, 1986; Bel l ows and Morse,
1993).
The al kal oi d fracti on of sabadi l l a seeds i s known to
contai n a mi xture of several compounds. Two esters of
veracevi ne, veratri di ne and cevadi ne (Fi gure 1), are
known to compri se more than 90% of the al kal oi d
fracti on of sabadi l l a (Hol an et al ., 1984), but rel ati vel y
l i ttl e attenti on has been focused on thei r separati on and
quanti fi cati on. Two reversed phase HPLC methods for
the separati on of cevadi ne and veratri di ne have been
publ i shed (Hol an et al ., 1984; Reed et al ., 1986), but both
methods suffer from poor resol uti on and excessi ve peak
tai l i ng.
As part of a study to better determi ne the rel ati ve
toxi ci ty of these al kal oi ds to ci trus thri ps, Scirtothrips
citri (Moul ton) (Thysanoptera: Thri pi dae), and the
range of natural vari ati on i n thei r concentrati on i n
commercial sabadilla formulations, a method was needed
to i sol ate rel ati vel y l arge quanti ti es of these al kal oi ds
for bi oassay. Because commerci al formul ati ons of sa-
badi l l a seeds are appl i ed for pest control i n aqueous
sol uti ons, part of the vari ati on i n the effecti veness of
commerci al sabadi l l a mi ght be due to parti al base
hydrol ysi s of the ester al kal oi ds i n al kal i ne wel l waters
pri or to appl i cati on. Thus, to test thi s hypothesi s, a
method al so was needed to moni tor the breakdown of
the esters to the l ess toxi c al kanol ami ne i n the envi ron-
ment.
Thi s study reports the i mprovement of a reversed
phase HPLC method to separate and quanti fy veratri -
di ne and cevadi ne by the addi ti on of a sol vent modi fi er.
Thi s general method al l owed preparati ve-scal e puri fi ca-
ti on of veratri di ne and cevadi ne from both veratri ne and
sabadi l l a seeds. I n addi ti on, wi th the i ncl usi on of a non-
ester al kal oi d as an i nternal standard, the method al so
al l owed the moni tori ng of the breakdown of veratri di ne
and cevadi ne i n aqueous sol uti ons over ti me as a
functi on of pH.
MATERI ALS AND METHODS
Sample Preparation. Veratri ne and veratri di ne (Si gma
Chemi cal Co.) were di ssol ved i n methanol (Fi sher Opti ma
grade) at 5 mg/mL and anal yzed by HPLC wi thout any
addi ti onal treatment because al l materi al di ssol ved i n the
methanol . Al kal oi ds were extracted from Veratran D (Dunhi l l
Chemi cal Corp., Rosemead, CA), a formul ati on of sabadi l l a
seeds, usi ng cl assi cal procedures for al kal oi d extracti on
(Hartmann, 1991). Bri efl y, 50 g of Veratran D was extracted
i n 500 mL of 5%(v:v) aceti c aci d i n water at room temperature
wi th sti rri ng for 1 h. The sol uti on was vacuum-fi l tered to
remove sol i d debri s. The fi l trate was basi fi ed wi th 4 N NaOH
to pH >10.0, and the al kal oi ds were extracted wi th three 500
mL porti ons of CH2Cl 2. The CH2Cl 2 sol uti on was concentrated
to dryness by rotary evaporati on, and the al kal oi ds were
redi ssol ved i n methanol for HPLC anal ysi s or preparati ve-
scal e HPLC. Veracevi ne was prepared from puri fi ed veratri -
di ne by methanol i c base hydrol ysi s fol l owi ng the procedures
of Pel l eti er and Jacobs (1953).
Figure 1. Structures of veratri di ne and cevadi ne.
149 J. Agric. Food Chem. 1996, 44, 149152
0021-8561/96/1444-0149$12.00/0 1996 American Chemical Society
Analytical HPLC. Chromatography was carri ed out wi th
a Beckman Model 332 chromatograph and an I SCO Model V4
vari abl e-wavel ength UV detector. I ni ti al HPLC condi ti ons
were those of Reed et al . (1986), usi ng vari ous modi fi cati ons
to thei r i socrati c mobi l e phase of methanol /0.1 M ammoni um
acetate, pH 5.5 (60:40). The mobi l e phase was fi l tered (0.22
m) and degassed wi th hel i um before use. Anal yti cal HPLC
was carri ed out usi ng a 4.6 mm 250 mm Beckman Ul tra-
sphere C18 col umn, 5 m parti cl e si ze, wi th a fl ow rate of 1.5
mL/mi n and effl uent moni tori ng at 245 nm. The i njecti on
vol ume was 20 L. El uates were col l ected wi th an automated
fracti on col l ector. The i denti ti es of veracevi ne, veratri di ne,
and cevadi ne were confi rmed by
1
H and
13
C nucl ear magneti c
resonance spectroscopy i n CDCl 3. I denti fi cati ons were made
on the basi s of the presence or absence of observed spectral
peaks i n each sampl e compared to previ ousl y publ i shed
spectral assi gnments (Kri shnamurthy and Casi da, 1988).
Solvent Modifiers for Improved Separation. The ad-
di ti on of a basi c modi fi er, such as tri ethyl ami ne, to the aqueous
phases of HPLC sol vents often i mproves the separati on of basi c
compounds, such as ami nes, on si l i ca-based col umns. The
addi ti on of an i on-pai ri ng agent to the mobi l e phase al so has
been reported to i mprove the separati on of i oni c compounds
such as al kanol ami nes (Stadal i us et al ., 1988; Szepesi , 1992).
The addi ti on of tri ethyl ami ne to the mobi l e phase i n concen-
trati ons up to 50 mM was i nvesti gated, as was the effect of
addi ng sodi um dodecyl sul fate (SDS) at 0.5 mM concentrati on
to the aqueous mobi l e phase. Vari ous modi fi cati ons to the
i oni c strength of the buffer and pH were made, but none
i mproved the separati on, and they wi l l not be reported i n
detai l . For thi s parti cul ar col umn, a pH of 4.5 for the
ammoni um acetate buffer was opti mal .
Preparative-ScaleHPLC. Preparati ve-scal e separati ons
of veratri di ne and cevadi ne from ei ther veratri ne or an extract
of Veratran D were carri ed out on a Phenomenex Parti ci l C8
col umn, 9.4 mm 500 mm, 10 m parti cl e si ze. The mobi l e
phase was a 70:30 mi xture of methanol /0.1 M ammoni um
acetate i n water to whi ch 0.5 mM SDS (see Resul ts and
Di scussi on) was added and the pH of whi ch had been adjusted
to 4.5 wi th aceti c aci d. The fl ow rate was 5 mL/mi n, the
i njecti on vol ume was 500 L, and the effl uent was moni tored
by UV detecti on at 245 nm.
Fracti ons were col l ected and concentrated by rotary evapo-
rati on to remove the methanol , and then the fracti ons were
basi fi ed wi th 4 N NaOH to pH >10.0. The puri fi ed al kal oi ds
were then separated from the resi dual SDS and ammoni um
acetate by extracti ng wi th three 250 mL porti ons of CH2Cl 2.
The CH2Cl 2 fracti ons were reduced i n vol ume by rotary
evaporati on, and then the fracti ons were transferred to
i ndi vi dual vi al s and taken to dryness by evaporati on under a
stream of N2. The dri ed al kal oi ds were stored at -20 C unti l
needed.
Evaluation of Potential Internal Standards. The non-
ester al kal oi ds papaveri ne and berberi ne were eval uated for
use as i nternal standards. An appropri ate i nternal standard
woul d be (1) sol ubl e i n di l ute aci d but extractabl e i nto organi c
sol vents under basi c condi ti ons (as are veratri di ne and ceva-
di ne), (2) chromatographabl e and detectabl e under our ana-
l yti cal condi ti ons but have a retenti on ti me di fferent from those
of veracevi ne, veratri di ne, cevadi ne, or any i mpuri ti es i n
sabadi l l a extracts, and (3) unaffected by prol onged exposure
to moderatel y basi c pH. Both papaveri ne and berberi ne were
extractabl e and chromatographabl e under the same condi ti ons
used for veratri di ne and cevadi ne. Berberi ne, however, showed
a degree of peak tai l i ng that woul d i nterfere wi th the mea-
surement of veratri di ne and was not i nvesti gated further.
Papaveri ne showed more promi se i n that i ts retenti on ti me
di ffered from al l other compounds of i nterest (see Resul ts and
Di scussi on). Three repl i cates of four papaveri ne standards
rangi ng i n concentrati on from 0.00195 to 0.0313 mg/mL were
prepared i n CH3OH. Standards were anal yzed by HPLC as
descri bed above to determi ne the l i neari ty of response of
papaveri ne and to devel op a cal i brati on curve.
The stabi l i ty of papaveri ne under exposure to al kal i ne
condi ti ons was determi ned as fol l ows. Dupl i cate 50 mL
sol uti ons of 0.0083 mg/mL of papaveri ne i n ammoni um acetate
(0.1 M, pH 4.5) were prepared. The pH of each was then
adjusted to 5.5 wi th NH4OH. The two sol uti ons then were
di vi ded i nto si x pai rs of al i quots of 8 mL each. No pH
adjustment was made to the pai r of sol uti ons assi gned to the
pH 5.5 treatment. The pH of the remai ni ng fi ve pai rs was
brought to 7.0, 8.5, 10.0, 11.5, or 13.0 wi th ei ther NH4OH (pH
7-10) or NaOH (pH 11.5 and 13). Al l sampl es were kept at a
constant temperature of 25 C. The sampl es were pl aced on
a shaker and removed 24 h l ater.
Sampl e workup was conducted as fol l ows: For the treat-
ments at pH e10, the pH was rai sed to >10 wi th NH4OH.
Each of the 12 sampl es was extracted wi th two 10 mL porti ons
of CH2Cl 2. The two CH2Cl 2 extracts per sampl e were combi ned
and concentrated by rotary evaporati on fol l owed by removal
of traces of sol vent under a stream of N2. The resi due was
redi ssol ved i n 2 mL of CH3OH and subjected to HPLC anal ysi s
as descri bed above.
The effl uent was moni tored at 245 nm, and the quanti ty of
papaveri ne i n each sampl e was determi ned from the cal i bra-
ti on curve (above). HPLC anal yses were run i n dupl i cate for
each sampl e. The 24 val ues (6 pH treatments 2 repl i cates
per treatment 2 anal ysi s per repl i cate) were subjected to
anal ysi s of vari ance to determi ne i f the quanti ti es of papav-
eri ne recovered from the sampl es after 24 h di ffered si gni fi -
cantl y from the i ni ti al quanti ty.
Quantification and Analyses of Breakdown of Ceva-
dine and Veratridine. An i nternal standard cal i brati on
tabl e was generated by performi ng HPLC anal ysi s on three
repl i cate cal i brati on mi xtures of papaveri ne at 0.024 mg/mL
and one of four concentrati ons each of veratri di ne and cevadi ne
i n 5% aceti c aci d and subjected to al l extracti on and workup
steps descri bed bel ow. The range of concentrati ons for ver-
atri di ne was from 0.03 to 1.00 mg/mL, and that for cevadi ne
was from 0.06 to 1.00 mg/mL.
For the breakdown studi es, dupl i cate mi xtures of 30 mg of
veratri di ne and cevadi ne each were di ssol ved i n 5 mL of 5%
aceti c aci d. Fi fty-fi ve mi l l i l i ters of ammoni um acetate (0.1 M,
pH 4.5) were then added to each mi xture. A 1.5 mL porti on
of a 1 mg/mL sol uti on of papaveri ne i n 5%aceti c aci d al so was
added as an i nternal standard to provi de a uni form i ni ti al
concentrati on of 0.024 mg/mL of papaveri ne i n al l sampl es.
The pH of both mi xtures was then adjusted to 5.5 wi th NH4-
OH. Each mi xture then was di vi ded i nto si x equal porti ons,
yi el di ng two mi xtures for each pH treatment. No pH adjust-
ment was made to the fi rst pai r. The pH of the remai ni ng
fi ve pai rs was brought to 7.0, 8.5, 10.0, 11.5, or 13.0 wi th ei ther
NH4OH or NaOH as above. Thi s caused the veratri di ne and
cevadi ne to preci pi tate and form a cl oudy suspensi on i n the
pH g10 treatments. Each of these si x pai rs of mi xtures was
di vi ded i nto fi ve 2 mL porti ons, one pai r for each sampl i ng
ti me, and pl aced i n a pol yethyl ene centri fuge tube. Care was
taken to ensure that al l sol i d materi al remai ned i n suspensi on
duri ng transfer by vortexi ng. The sampl es for ti me 0 were
i mmedi atel y extracted as descri bed bel ow. The remai ni ng
pai rs were pl aced on a shaker and removed 1, 2, 4, or 24 h
l ater. Al l sampl es were kept at a constant temperature of 25
C.
Sampl e workup was conducted as fol l ows: For the sampl es
at pH e10, the pH was rai sed to >10 wi th NH4OH. Al kal oi ds
were then extracted wi th si x 10 mL porti ons of CH2Cl 2, and
the CH2Cl 2 extracts per sampl e were combi ned and concen-
trated and then dri ed under N2 as descri bed above. The
al kal oi ds were redi ssol ved i n 2 mL of CH3OH and subjected
to HPLC anal ysi s. Quanti ti es of veratri di ne and cevadi ne
recovered were cal cul ated rel ati ve to the i nternal standard,
and the percent l oss of each al kal oi d over ti me was then
cal cul ated as a functi on of pH. The mean percent recoveri es
(( standard errors of the mean) of veratri di ne and cevadi ne
are reported.
RESULTS AND DI SCUSSI ON
Absorbance of UV l i ght i n the 240-260 nm regi on i s
due to the presence of veratri c aci d or angel i c aci d
esteri fi ed wi th veracevi ne. Veracevi ne i tsel f has com-
150 J. Agric. Food Chem., Vol. 44, No. 1, 1996 Hare
parati vel y l i ttl e UV absorbance i n thi s regi on. The use
of 245 nm for detecti on fol l owed the methods of Hol an
et al . (1984) and was a compromi se between opti mal
detectabi l i ty of al l compounds and opti mal si gnal to
noi se rati o i n our system. Refracti ve i ndex detecti on
has al so been used wi th these compounds (Hol an et al .,
1984). However, i n my study, there was a consi derabl e
l oss of sensi ti vi ty and an i ncrease i n the basel i ne noi se
when refracti ve i ndex detecti on was eval uated i n tan-
dem wi th UV detecti on. Refracti ve i ndex detecti on
therefore was deemed to be unsui tabl e for quanti fi cati on
of veratrum al kal oi ds i n thi s study.
Solvent Modifiers for Improved Separation.
Wi thout any sol vent modi fi ers, cevadi ne, especi al l y,
exhi bi ted substanti al peak tai l i ng on both the C
18
anal yti cal col umn (Fi gure 2A) and the C
8
preparati ve
col umn (Fi gure 2C). Tri ethyl ami ne at concentrati ons
up to 50 mM offered no i mprovement i n peak separati on
or resol uti on. The addi ti on of SDS at 0.5 mM to the
aqueous mobi l e phase reduced substanti al l y the tai l i ng
of cevadi ne (Fi gure 2B) and al l owed for basel i ne separa-
ti on of veratri di ne from cevadi ne on the preparati ve
col umn (Fi gure 2D). SDS at 0.5 mM therefore was
added routi nel y to the ammoni um acetate mobi l e phase
i n al l subsequent studi es.
Evaluationof Potential Internal Standards. The
retenti on ti me of papaveri ne fel l conveni entl y between
those of veracevi ne and veratri di ne (Fi gure 3). Peak
areas of papaveri ne i ncreased l i nearl y wi th concentra-
ti on over the range between 0.00195 and 0.0313 mg/
mL (r
2
) 0.996, four concentrati ons anal yzed i n tri pl i -
cate). The l i mi t of detecti on (three ti mes si gnal noi se)
was 0.00025 mg/mL. No si gni fi cant breakdown of
papaveri ne was observed after exposure to any pH from
5.5 to 13 for 24 h (Tabl e 1).
Quantification and Analyses of Breakdown of
Cevadine and Veratridine. The peak area of ver-
atri di ne i ncreased l i nearl y wi th concentrati on between
0.03 and 1.00 mg/mL (r
2
) 0.994), and the peak area of
cevadi ne i ncreased l i nearl y wi th concentrati on between
0.06 and 1.00 mg/mL (r
2
) 0.992). Li mi ts of detecti on
(three ti mes si gnal noi se) were 0.0076 mg/mL for
veratri di ne and 0.018 mg/mL for cevadi ne.
No si gni fi cant breakdown of ei ther cevadi ne or ver-
atri di ne was observed over 24 h at pH e10 (Fi gure 4).
Because i t i s unl i kel y ei ther that the water used for
pesti ci de appl i cati on i s as basi c as thi s or that the
pesti ci de i s l eft i n the sprayer for more than 1 day,
commerci al pesti ci de formul ati ons of sabadi l l a al kal oi ds
are unl i kel y to be hydrol yzed pri or to appl i cati on.
Hi gher, al bei t envi ronmental l y unreal i sti c, pH val ues
were i ncl uded i n thi s experi ment si mpl y to better
Figure 2. HPLC chromatograms of veratri ne al kal oi ds: (A
and B) veratri ne wi thout and wi th a sol vent modi fer on a 4.6
mm 250 mm Beckman Ul trasphere C18 col umn, 5 m
parti cl e si ze, fl ow rate 1.5 mL/mi n; (C and D) Veratran D
wi thout and wi th a sol vent modi fi er on a 9.4 mm 500 mm
Phenomenex Parti ci l C8 col umn, 10 m parti cl e si ze, fl ow rate
5 mL/mi n. El uents: (A) 60%methanol , 40%0.1 M ammoni um
acetate, pH 4.5; (B) 60% methanol , 40% 0.1 M ammoni um
acetate + 0.5 mM SDS, pH 4.5; (C) 70% methanol , 30% 0.1 M
ammoni um acetate, pH 4.5; (D) 70% methanol , 30% 0.1 M
ammoni um acetate + 0.5 mM SDS, pH 4.5. Detecti on for al l
was by UV at 245 nm.
Figure 3. Chromatography of veracevi ne, veratri di ne, and
cevadi ne wi th SDS and wi th papaveri ne as an i nternal
standard for quanti tati on. Condi ti ons were the same as i n
Fi gure 2B.
Table 1. Recovery of Papaverine after 24 h at Different
pH Levels
pH
recovery of
papaveri ne
a
(%) pH
recovery of
papaveri ne
a
(%)
5.5 98.6 ( 0.03 10.0 96.9 ( 0.89
7.0 103.8 ( 3.64 11.5 100.7 ( 0.50
8.5 99.9 ( 0.99 13.0 99.0 ( 0.05
a
Mean ( standard error of the mean for dupl i cate sampl es.
Figure 4. Mean (( standard error) percent recovery of
veratri di ne (top) and cevadi ne (bottom) i n aqueous sol uti ons
at 25 C as a functi on of pH and ti me: (b) pH 5.5; (O) pH 7.0;
(9) pH 8.5; (0) pH 10.0; (1) pH 11.5; (3) pH 13.0.
HPLC of Veratridine and Cevadine J. Agric. Food Chem., Vol. 44, No. 1, 1996 151
understand the sensi ti vi ty of cevadi ne and veratri di ne
to hydrol ysi s under basi c condi ti ons. At a pH of 11.5,
45.9% of the veratri di ne was l ost after 4 h but 47.6% of
the cevadi ne remai ned after 24 h. Both al kal oi ds were
80-90% degraded wi thi n 1 h at pH 13 and compl etel y
degraded after 24 h. Thus, whi l e both veratri di ne and
cevadi ne can be degraded by hi gh pH, such hi gh pH
val ues are unl i kel y to be encountered duri ng the process
of mi xi ng and appl yi ng commerci al sabadi l l a pesti ci de
formul ati ons to crops. Thi s HPLC method al so may
provi de the basi s for techni ques to moni tor the break-
down of veratri di ne and cevadi ne to thei r rel ati vel y l ess
toxi c precursors (Al l en et al .; 1945, I kawa et al ., 1945;
Bergmann et al ., 1958) i n the envi ronment after ap-
pl i cati on.
ACKNOWLEDGMENT
I thank J. Kushner and E. W. McCol l um for techni cal
assi stance, S. Mi dl and for NMR spectroscopy anal yses,
and J. G. Mi l l ar for comments on a previ ous draft.
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Recei ved for revi ew December 5, 1994. Revi sed manuscri pt
recei ved May 9, 1995. Accepted October 11, 1995.
X
Thi s
research was funded by grants from the Cal i forni a Ci trus
Research Board and from Dunhi l l Chemi cal Corp.
JF9406828
X
Abstract publ i shed i n AdvanceACS Abstracts, De-
cember 1, 1995.
152 J. Agric. Food Chem., Vol. 44, No. 1, 1996 Hare