Quantitative GC analysis of secondary alcohol pheromones: Determination of release rate of red palm weevil, Rhynchophorus ferrugineus, pheromone from lures

Article abstract of DOI:10.1023/A:1021057501459

Aliphatic secondary alcohols are components of several aggregation pheromones of important beetle and weevil pests. Some of these pheromones are used frequently for the monitoring and mass trapping of the relevant insects. We encountered severe difficulties in direct GC quantitative analysis of these compounds. Therefore, we developed a simple GC analysis of secondary alcohols converting them to trifluoroacetyl derivatives and using secondary alcohol acetates as internal standards. This method was applied for the quantitative analysis of several secondary alcohols, including the aggregation pheromone components of the almond bark beetle and the red palm weevil. The release rate of the latter pheromone from commercial lures was also determined.

Full text of DOI:10.1023/A:1021057501459

C
Journal of Chemical Ecology, Vol. 28, No. 11, November 2002 ( 2002)
QUANTITATIVE GC ANALYSIS OF SECONDARY ALCOHOL
PHEROMONES: DETERMINATION OF RELEASE RATE OF
RED PALM WEEVIL, Rhynchophorus ferrugineus,
PHEROMONE FROM LURES
A. ZADA,1, V. SOROKER,1 M. HAREL,1 J. NAKACHE,2
and E. DUNKELBLUM1
1Institute of Plant Protection
The Volcani Center
Bet-Dagan, 50250, Israel
2Eden Experiment Station
Bet Shean, Israel
(Received January 10, 2002; accepted July 5, 2002)
Abstract—Aliphatic secondary alcohols are components of several aggregation
pheromones of important beetle and weevil pests. Some of these pheromones
are used frequently for the monitoring and mass trapping of the relevant in-
sects. We encountered severe difficulties in direct GC quantitative analysis of
these compounds. Therefore, we developed a simple GC analysis of secondary
alcohols converting them to trifluoroacetyl derivatives and using secondary alco-
hol acetates as internal standards. This method was applied for the quantitative
analysis of several secondary alcohols, including the aggregation pheromone
components of the almond bark beetle and the red palm weevil. The release rate
of the latter pheromone from commercial lures was also determined.
KeyWords—Aggregationpheromones, secondaryalcohols, trifluoroacetylderiva-
tives, GC analysis.
INTRODUCTION
Several aliphatic secondary alcohols are common components of aggregation
pheromones of weevils and beetles (Bartelt, 1999; Schlyter and Birgersson, 1999).
The use of pheromone lures in pest management requires a quantitative determi-
nation of the content in order to measure the release rate of the pheromone and the
longevity of the lures. Capillary GC analysis, with an internal standard, is the best
To whom correspondence should be addressed. E-mail: anatzada@volcani.agri.gov.il
2299
C
0098-0331/02/1100-2299/0 2002 Plenum Publishing Corporation

2300
ZADA, SOROKER, HAREL, NAKACHE, AND DUNKELBLUM
method for quantitation. However, peaks of secondary alcohols have a tendency
to broaden and tail on different capillary columns, which makes their direct anal-
ysis difficult and inaccurate. The problem is particularly severe when analyses are
performed repeatedly on the same column.
In this paper, we present a simple GC method of quantitation of secondary
alcohols using their trifluoroacetyl derivatives and appropriate acetates as inter-
nal standards. The effect of the mode of injection on the analysis has also been
studied. The following alcohols were analyzed: 4-methyl-5-nonanol, 4-methyl-3-
heptanol, 4-methyl-3-hexanol, and 2-octanol. These alcohols (except 2-octanol)
are pheromone components of two agricultural pest insects, the red palm wee-
vil (Rhynchophorus ferrugineus) (Perez et al., 1996) and the almond bark beetle
(Scolytus amygdali) (Ben-Yehuda et al., 2000), respectively.
METHODS AND MATERIALS
Chemicals. Mixtures of syn/anti 4-methyl-3-hexanol and 4-methyl-5-nona-
nol were prepared by Grignard reaction (Scheme 1) from 2-bromobutane and pro-
pionaldehyde and from 2-bromopentane and valeraldehyde, respectively, using
magnesium turnings in THF (dried over Na + benzophenone). Distillation was
carried out under reduced pressure (142 C/760 mmHg and 87 C/12 mmHg, re-
spectively) to obtain the pure alcohols (97–100% by GC). The acetate derivatives
were prepared from the corresponding alcohols and acetic anhydride in pyridine
and purified by chromatography on SiO2, using hexane and 1% ether as eluent. All
other chemicals, including syn/anti (60:40) 4-methyl-3-heptanol, were purchased
from Aldrich.
SCHEME 1.
Gas Chromatography. Analyses were performed on an HP6890 gas chro-
matograph equipped with an HP5 column (30 m 0.25 mm ID; 0.25 m), flame

QUANTITATIVE GC ANALYSIS OF SECONDARY ALCOHOL PHEROMONES
2301
ionization detector, and a split/splitless injector. Helium was used as the carrier
gas with a flow rate of 1.5 ml/min. The analysis was performed in the split mode
with a ratio of 10:1. The column was kept at 50 C for 3 min and then programmed
at a rate of 20 C/min to 100 C for the analysis of 4-methyl-3-hexanol, 4-methyl-
3-heptanol, and 2-octanol and kept at 60 C for 2 min, and then programmed at a
rate of 20 C/min to 130 C for the analysis of 4-methyl-5-nonanol. The injector
and detector were maintained at 220 C.
Procedure of Analyses. Solutions of 4-methyl-5-nonanol, 4-methyl-3-hepta-
nol, 4-methyl-3-hexanol, and 2-octanol were prepared in hexane at a concentration
of 1 mg/ml ( = 1 g/ l). Representative aliquots of each solution (1, 5, 10, 50,
75, 100, 150, and 200 l) were placed in a 4-ml vial containing 1 ml of hexane.
Then, an appropriate amount of trifluoroacetic anhydride (50–100 l) was added,
and the vial was shaken thoroughly. The reaction mixture was shaken occasionally
for 1 hr at room temperature and then cooled in an ice-bath. The derivatization
was terminated by addition of 1 ml of 5% NH4OH (aq) and 100 l of a hexane
solution containing 100 g of the appropriate acetate derivative (stock solution of 1
mg/ml). Fifty micrograms of the acetates’ internal standard was used for the small
amounts of analyzed alcohols (1–10 g). The mixture was shaken thoroughly, and
the organic phase was separated with a pipet. Aliquots of approximately 2 l of
the organic phase were injected (three replicates) into the gas chromatograph. A
representative analysis of 100 g of 4-methyl-5-nonanol with 50, 75, and 100
g
of the corresponding acetate as internal standard was injected several times and
gave the same results.
Application of the Method with Red Palm Weevil Pheromone Lures. Phero-
mone lures of the red palm weevil were purchased from ChemTica (Costa Rica).
They contained 750 5 mg pheromone of which 90% is 4-methyl-5-nonanol and
10% is 4-methyl-5-nonanone enclosed in a plastic bag with a membrane (Hallett
et al., 1999). The lures were placed in weevil bucket traps in a date plantation in
the Eden ranch, Jordan Valley. The traps also contained a mixture of plant material
(molasses sugar and dates) in water and a bottle of ethyl acetate. Every 10 d,
three lures were removed from the orchard and kept at 4 C until analyzed. The
plastic bag containing the pheromone was cut into pieces and immersed in 20 ml
of dichloromethane (CH2Cl2) to extract the pheromone. The samples were kept in
an ultrasound bath for 30 min and left overnight at 4 C. The plastic material was
not affected by the solvent. For GC analysis, samples were diluted by a factor of
100. Then, an aliquot of 100 l was further diluted with 1 ml hexane and 50 l of
the appropriate acetate standard were added.
RESULTS AND DISCUSSION
It is well known that quantitative GC analysis of secondary alcohols is dif-
ficult and inaccurate due to peak broadening and tailing (Krupcik et al., 1975).

2302
ZADA, SOROKER, HAREL, NAKACHE, AND DUNKELBLUM
FIG. 1. GC profiles of 2-octanol versus octyl-2-trifluoroacetate. HP5 column was kept
at 50 C for 3 min and then programmed at a rate of 20 /min to 100 C. Split ratio 10:1.
Despite the availability of improved capillary GC columns today, secondary al-
cohols display erratic behavior, particularly with aging columns. The columns
are too expensive to be replaced after a short usage. Periodical weighing has
been recommended as an alternative method in order to determine release rates
of such pheromones from lures (Vickers et al., 1998; A.C. Oehschlager, per-
sonal communication). However, loss of weight is not specific to the loss of
the pheromone alone. In addition, it gives erratic results due to absorption of
dust and moisture. GC–FID analysis, with an appropriate internal standard, is
the most accurate method, provided that peaks are sharp and the analysis is
consistent.
During our work on mass trapping of the red palm weevil and the almond
bark beetle with pheromone lures, we were in need of a reliable quantitative anal-
ysis of their respective main pheromone components, 4-methyl-5-nonanol and
4-methyl-3-heptanol. Direct GC analysis of the alcohol gave inaccurate results
and, therefore, we looked for an alternative route and decided to use a deriva-
tization method. We chose trifluoroacetylation because this derivatization of al-
cohols is easy to perform. It is quantitative and the formed trifluoroacetates can
be readily analyzed by capillary GC-FID (Krupcik et al., 1994). Peaks are sym-
metrical and sharp (Figure 1). Therefore, sensitivity is higher as compared with
the parent alcohols. The method has been employed for the analysis of alco-
hols and sugars (Krupcik et al., 1994; Hisatomi et al., 2000). However, as far
as we know, trifluoroacetylation has not been used for quantitative analysis of
secondary alcohols by GC. The FID response of the acetates and the trifluoroac-
etates is practically identical (GC-FID factor >0.98), and the latter always elute
before their corresponding acetates (Representative example is in Scheme 2 and
Figure 2).

QUANTITATIVE GC ANALYSIS OF SECONDARY ALCOHOL PHEROMONES
2303
SCHEME 2.
The method was tested with various amounts of 4-methyl-3-hexanol,
4-methyl-3-heptanol, 2-octanol, and 4-methyl-5-nonanol in the range of 1–200 g.
Representative results of 4-methyl-3-heptanol and 4-methyl-5-nonanol, presented
in Figures 3 and 4, indicate an excellent correlation (>99%) between the amount
of secondary alcohol inserted and detected by the GC method. The use of the
acetate of the same alcohol as the internal standard is not essential. For example,
4-methyl-3-hexanol and 2-octanol were analyzed with 4-methyl-3-heptylacetate
as the internal standard, and the results were accurate. For better peak shape and
resolution, we recommend performing the GC analysis in the split mode and pro-
graming the oven temperature so that both the analyte and the standard will be
analyzed isothermally. The trifluoroacetylation reaction is quantitative, and no
FIG. 2. GC profiles of the analysis of syn/anti (60:40) 4-methyl-3-heptanol with its acetate
derivative as internal standard versus analysis of 4-methyl-3-heptyltrifluoroacetate using
the same standard. HP5 column was kept at 50 C for 3 min and then programmed at a rate
of 20 /min to 100 C. Split ratio 10:1

2304
ZADA, SOROKER, HAREL, NAKACHE, AND DUNKELBLUM
FIG. 3. GC quantitative analysis of 4-methyl-3-heptanol. Each point is the average of
three anlyses.
residual alcohol was detected by GC in all cases. The use of varying amounts of
alcohols or internal standard does not introduce any significant error, as shown by
the excellent reproducibility.
The red palm weevil, Rhynchophorus ferrugineus (RPW), is a serious pest
of dates. The major component of its aggregation pheromone is (S,S)-4-methyl-
5-nonanol (Perez et al., 1996). Commercial lures, containing racemic syn/anti
4-methyl-5-nonanol, are being used in the Far and Middle East for mass trapping
(Hallett et al., 1999; Vidyasagar et al., 2000). The determination of the release rate
of the pheromone is essential in order to evaluate the longevity of the lures. Direct
analysis of 4-methyl-5-nonanol produced erratic results. We applied our method
FIG. 4. GC quantitative analysis of 4-methyl-5-nonanol. Each point is the average of
three anlyses.

QUANTITATIVE GC ANALYSIS OF SECONDARY ALCOHOL PHEROMONES
2305
FIG. 5. Release rate of 4-methyl-5-nonanol from the ChemTica lures under field conditions.
and obtained consistent results. The behavior of the lure for the whole period
can be represented by an exponential curve (Figure 5). However, the release rate
of about 8 mg/day is linear for 58 days, and then the release rate decreases to
3–4 mg/day for another 40 days (Figures 5 and 6). This behavior corresponds with
the manufacturer’s specifications (release rate of 3–10 mg/day) and the temperature
dependence of the release rate. The average temperatures in the plantation for the
first period (0–58 d) of the field test was 23.5–36.6 C, and of the second one
(58–98 d) was 17–29.8 C.
In conclusion, the quantitation of secondary alcohol aggregation pheromones
is of practical importance and is not merely an analytical exercise. Several beetle
and weevil pheromones containing such components are being used for mass
trapping in Integrated Post Management programs. The method presented in this
study will facilitate the evaluation of such lures and will enable the determination
of their longevity and release rate profiles.
FIG. 6. Release rate of 4-methyl-5-nonanol from the ChemTica lures for the first period of
58 d of the field test.

2306
ZADA, SOROKER, HAREL, NAKACHE, AND DUNKELBLUM
Acknowledgments—We thank the Paul Vermesh family for the scholarship supporting Dr. Anat
Zada. This study was supported by Chief Scientist Grant No. 137-1127-1-61 of the Israeli Ministry of
Agricultural.
REFERENCES
BARTELT, R. J. 1999. Weevils, Pheromones of Non-Lepidopteran Insects Associated with Agricultural
Plants, pp. 91–112, in J. Hardie, and A. K. Minks (eds.). CAB International, Oxford, United
Kingdom.
BEN-YEHUDA, S., TOLASCH, T., FRANCKE, W., GRIES, R., GRIES, G., DUNKELBLUM, E., and MENDEL, Z.
2000. Aggregation pheromone of the almond bark beetle Scolytus amygdali (Coleoptera: Scolyti-
dae). Book of Abstracts, 25th IOBC-WPRS Meeting, Samos, Greece.
HALLETT, R. H., OEHLSCHLAGER, A. C., and BORDEN, J. H. 1999. Pheromone trapping protocol for the
Asian palm weevil, Rhynchophorus ferrugineus (Coleoptera: Curculionidae), Int. J. Pest Manage.
45:231–237.
HISATOMI, E., MATSUI, M., KOBAYASHI, A., and KUBOTA, K. 2000. Antioxidative activity in the
pericarpandseedofJapanesepepper(XanthoxylumpiperitumDC). J. AgricFoodChem. 48:4924–
4928.
KRUPCIK, J., TESARIK, K., and HRIVNAK, J. 1975. Separation of secondary alcohols by capillary gas
chromatography. Chromatographia 8:553–558.
KRUPCIK, J., BENICKA, E., MAJEK, P., SKACANI, I., and SANDRA, P. 1994. Relationship between struc-
ture and chromatographic behavior of secondary alcohols and their derivatives separated by high-
resolution gas chromatography with a modofied -cyclodextrin stationary phase. J. Chromatogr.
A 665:175–184.
SCHLYTER F. and BIRGERSSON, G. A. 1999. Forest Beetles, pp. 113–148, in J. Hardie and A. K.
Minks (eds.). Pheromones of Non-Lepidopteran Insects Associated with Agricultural plants,.
CAB International, Oxford, UK.
PEREZ, A. L., HALLET, R. H., GRIES, R., GRIES, G., OEHLSCHLAGER, C., and BORDEN, J. H. 1996.
Pheromone chirality of asian palm weevils, Rhynchophorus ferrugineus, (Oliv.) and R. vulneratus
(Panz.) (Coleoptera: Curculionidae). J. Chem. Ecol. 22:357–368.
VICKERS, R. A., THWAITE, W. G., WILLIAMS, D. G., and NICHOLAS, A. H. 1998. Control of codling
moth in small plots by mating disruption: alone and with limited insecticide, Entomol. Exp. Appl.
86:229–239.
VIDYASAGAR, P. S. V., HAGI, M., ABOZUHAIRAH, R. A., MOHANA, O. A. E., and SAIHATI, A. A. 2000.
Impact of mass pheromone trapping on red palm weevil: Adult population and infestation level
in date palm gardens of Saudi Arabia. The Planter 76:347–355.