Optimized Gas Chromatographic Analysis of Natural

Pyrethrins and Pyrethroids

Thomas J. Class
Department of Analytical and Environmental Chemistry at the University of Ulm, W-7900 Ulm, FRG

Key Words:
Gas chromatograpy, GC
Analysis
Pyrethrins
Pyrethroids
Insecticide formulations

Summary
Gas chromatographic analysis of natural pyrethrins in insecticidal pyrethrum extract and in insecticide formulations is
achieved by on-column injection with hydrogen as carrier gas
under constant flow and programmed pressure conditions.
Short thin-film fused silica capillaries (DB 5 and DB 1701)
coupled to retention gaps separate the pyrethrins (cinerin Iand
II, jasmolin I and 1,and pyrethrin I and 11) and their analogue
S-bioallethrin (Esbiol) in less than twelve minutes at temperatures below 210 "C without any discrimination, degradation or
isomerization of thermally labile pyrethrin I and II. With Sbioallethrin added as internal standard, spray and powder
formulations are extracted with ethyl acetate and analyzed by
gas chromatography with flame ionization detection for pyrethrum constituents, for other pyrethroids such as tetramethrin,
cyphenothrin, cypermethrin, and for the commonly used synergist piperonyl butoxide.

S-bioallethrin (A) : R 1 = CH3, R z = H

Pyrethrins

chrysarithemates (I)

pyrethrates (11)

cinerin ic).
jasmolin (J):
pyrethrin (PI-

CI R ~ = C I S , R ~ = C H ~ C I I : R ~ = C O ~ C H ~ , R Z = C H ~
J I . R ~ = C H ~ , R Z = C H ~ CJ IHI ~: R ~ = C O ~ C H ~ , R Z = C H ~ C H ~
PI R1=CH3,R2=CH=CH2 PII:R1=COZCH3,R2=CH=CH2

1 Introduction
Chromatographic analysis of the six natural pyIethrins cinerin I
and I1 (CI and CLI),jasmolin I and I1 (JI and JII), and pyrethrin I
and I1 (PI and PII) in insecticidal pyrethrum extract from the
daisy -like flower Chrysanthemum cinemiifohm [ 11and in insecticide formulations is generally done either by liquid chromatography (LC)[a-41, or by gas chromatography (GC) with packed or
capillary columns [5-13jI or by supercritical fluid chromatography
(SFC) with infrared detection 1141. By means of GC-mass spectrometry (MS) Holmsted and Soderlund [5] observed quite early
that PI and PI1 with their cis-pentadienyl moiety undergo thermal
isomerization to form iso-PI and iso-PI1 at injection and column
temperatures above 210 "C as proposed by Elliott 1151.
In the present study GC analysis of the pyrethrins is optimized by
avoiding any thermal effects during injection and separation. This
is achieved by cool on-column injection with hydrogen at fast
carrier gas velocities under constant flow or programmed pressure conditions using short thin-film capillaries. Thus even the
latest eluting pyrethrin PI1 is separated at temperatures below
210 "C(Table1).This is demonstrated for pyrethrum extract and
for insecticidal formulations which are intended for household
and garden use and contain pyrethrins, the pyrethroids tetramethrin (T),
cyphenothrin (Cyph)and cypermethrin (Cy),and the
commonly used synergist piperonyl butoxide (PB).

48

Journal of High Resolution Chromatography

PI, PI1

iso-PI, iso-PI1

if

'R2

cyphenothrin (Cyph): R 1 = CH3, R Z =

HC

CN
cyperrnethrin (Cy) :

R1 =

CI, R,

0 1991 Dr.Alfred Huethig Publishers

Optimized GC of Pyrethrins and Pyrethroids

piperonyl butoxide (PB) :

lee1A

80
2 Experimental
Pyrethrum extract (47 %) and S-bioallethrin (Esbiol, PynaminB
forte, A) were obtained from Riedel-de-Haen; tetramethrin (Neopynamin@,T),cypermethrin (Cy), and piperonyl butoxide (PB)
originated from Promocheni, cyphenothrin (Cyph)was a gift from
the Thompson-Siege1 GmbH. Individual pyrethrins were isolated
from pyrethrum extract by preparative thin-layer chromatography
(TLC) and normal phase liquid chromatograpy (NP-HPLC) on
Nucleosil-N02 [ 16, 171. Several insecticidal formulations such as
powder or sprays were bought in drugstores.
Gas chromatography-mass spectrometry (GC-MS)employed a HP
5980 gas chromatograph equipped with splitisplitless injector
(200 "C) and with a J&W DB5 (Promocheni, FRG) fused silica
capillary (25 m length, 0.25 mm i.d., 0.1 Iim film) (column 1)
directly interfaced to the VG TS 250 mass spectrometer. Helium
was used as carrier gas at 7 psi (carrier gas velocity about 45 cm/s
at 90 "C)with the following temperature program: 90 "C for 3 min,
30"imin to 180 "C,then with 3"/min to 240 "C. Electron impact
(70 eV) ionization (EI) gave small but detectable molecular ions
and characteristic fragmentation patterns for the pyrethrins [18].
Chemical ionization (CI) with methane as reactant gas provided
additional molecular weight information by the presence of the
corresponding [M+H]+ions.
High resolution gas chromatography (HRGC) utilized a Hewlett
Packard 5980 Series I1 gas chromatograph equipped with a
split/splitless and a programmable pressure cool on-column
injector, and with flame ionization (FID) and electron capture
(ECD)detectors. Separations were performed on a J&W DB5 fused
silica capillary (10 m length, 0.25 mm i.d., 0.1 pm film) (column 2)
and on a J&W DB1701 fused silica capillary (12 m length, 0.25 mm
i d . , 0.15pm film) (column 3) both connected to retention gaps
(1.5 m length, 0.32 mm i.d., uncoated and deactivated, Chrompack, The Netherlands). Hydrogen was used as carrier gas under
constant flow (8 psi at 90 "C,about 80 cmis carrier gas velocity,
column 2) or programmed pressure (10 psi, 1 min, then with
20 psi/min to 30 psi corresponding to about 100 cm/s carrier gas
velocity, column 3) conditions with on-column injection. The
temperature program was as follows: 90 "C, 1 min, with 30"imin to
180 "C. then with 3"/mln to 220 "C.
Insecticidal formulations (e.g. 0.1 or 1 ml spray or 0.5 g powder)
were extracted with 2 x 2 mi ethyl acetate after spiking w-ith an
internal standard (S-bioallethrin, 2 ngiul, 20 ~t1in ethyl acetate).
The extracts were diluted if necessary and analyzed by GC with
FID detection resulting in a reproducibility of k 15 % or better for
three different extractions.

78

ISO-PI1
1891

HRGC-MS (El) total ion chromatogram (splitless injection, column I)

of pyrethrum extract (1 pg/pI, 0.5 pl) with thermal isomerization
products of PI and PI1 formed in the hot (200 "C) injector and on the
column.

these regions is caused by thermal isomerization of the cispentadienyl moieties in injector and on the column at elution
temperatures above 200 "Cfor PI and PI1 (Table 1).
CI-MS with the VG TS 250 gave intense fragmentation, very low
intensity for the quasi-molecular ion [M+H]+ and no M+29 or
M+41 ions for the pyrethrins and the pyrethroids. This stands in
contrast to a GC-MS (CI) study on pyrethroid metabolites [17]
performed on a Hewlett-Packard 5985 GC-MS system where the
presence of intense M+1, M+29, and M+41 ions was used for the
identification of parent pyrethroids, metabolites and the corresponding diazomethane and trimethylsilyl derivatives.
The elution temperatures of the pyrethroids are reduced well
below 200 "C by the use of hydrogen as carrier gas with its flat van

Table 1
GC elution temperatures (%) of thermolabile PI and PI1 at
different GC conditions.
___

Problems arising in GC analysis of pyrethrins are illustrated in

Figure 1by the GC-MS (EI) total ion chromatogram of pyrethrum
extract. EI mass spectra recorded between spectia no. 532 (PI)
and 652 (iso-PI) all have molecular ions at 328 m/e, whereas
spectra no. 950 (PII) to 1091 (iso-PII) feature molecular ions at
372 m/e. The resulting shape of the total ion chromatogram in
Journal of High Resolution Chromatography

cam-

column 1 column 2 column 3 Class et al., 1990

DB5,H2 DB1701,H2 SPB5a),H2SPB5bl,H2
pound DB5,He
PI
PI1
-

3 Results and Discussion

MIN

Figure 1

207
229

184
195

190
209

185
197

205
226

Splitless injection onto a Supelco SPB5 fused silica capillary column (15 m
length, 0.32 mm i.d., 0.25 pmfilm) with hydrogen as carrier gas (8psi)and
ECD detection. Temperature program: 90 "Cfor 2 min. 30"/min to 180 "C,
then with 3"imin to 240 "C.A modified temperature program (as above
but from 150 "C with 2"imin to 200 "C) was used in a special study
involving only chrysanthemates and resulted in an elution temperature
for PI of 164 "C.
Splitless injection onto a Supelco SPB5 fused silica capillary column (30m
length, 0.25 mm i.d., 0.25pm film) with hydrogen as carrier gas (30 psi)
and CI-MS deteclion temperature program as ind).
VOL. 14, JANUARY 1991

49

Optimized GC of Pyrethrins and Pyrethroids

Deemter curve which allows carrier gas velocities of 80 cm/s and

more without significant loss of efficiency. Short thin-film
columns provide enough resolution for the separation of the
natural pyrethrins without the necessity of increased elution
temperatures. This was demonstrated recently in a study on the
microsomal oxidase metabolites of S-bioallethrin and the pyrethrins [17] employing GC-ECD and GC-MS with chemical ionization (CI) (Table 1). Nevertheless, splitless injection discriminates
the pyrethrates CII, JII and PI1 and some isomerization still
occurs.
Optimized GC analysis of the pyrethrins in pyrethrum extract
(Figure 2) and in a standard solution containing equal amounts
of the six individually isolated pyrethrins, S-bioallethrin, tetramethrin, cypermethrin and piperonyl butoxide (Figures 3a and
b) employs the 0.25 mm i.d. capillary (column 2) attached to a
separates into two
0.25 mm 1.d. retention gap. Tetramethrin (T)
GC peaks which on GC-MS (EI) analysis feature identical mass
spectra and are tentatively assigned to the isomers corresponding
to cis- and trans-chrysanthemate in analogy to the elution order of
cis- and trans-allethrin where an authentic trans-isomer (Sbioallethrin) is available. The cluster of three distinct peaks
originating from cypermethrin (Cy) indicates the presence of all
eight possible stereoisomers. This was confirmed by further
optimization of the gas chromatographic analysis which resulted
in four well resolved GC peaks with identical mass spectra thus
indicating the diastereomeric resolution into four pairs of enantiomeres.

With column 3 and its slightly more polar stationary phase the
pyrethrins elute at higher elution temperatures under constant
carrier gas flow conditions, and thermal degradation occurs. As
resolution and selectivity are enhanced with column 3, a pressure
program starting at a relatively low hydrogen pressure (10 psi) to
avoid problems with manual on-column injection, followed by a
sharp pressure increase to 30 psi, causes higher carrier gas flow
with only little loss of separation efficiency (Figure 4). PI1 still
elutes at higher temperature than on column 2 (Table l ) ,but no
thermal isomerization is observed. Carrier gas pressures above
30 psi further reduce the retention times of the pyrethrates.
However peak broadening indicates non-ideal flow conditions
and loss of chromatographic efficiency. The order of elution of the
pyrethrins on column 2 is maintained on column 3 but the relative
positions of some impurities present in the solution are varied.

Figure 5 shows a chromatogram obtained by extraction of an

insecticidal formulation to which S-bioallethrin was added as
internal standard. Recoveries of internal standard, pyrethrum and
individual pyrethroids were always better than 80 %. Four different sprays and one powder formulation were analyzed. Spray no. 1
and another one, no. 2 (with different brand name but similar
chromatographic pattern and comparable concentrations) contain pyrethrum extract (0.2 mg/ml) together with the synergist

a
\

CI

The incorporation of the retention gap into the separation system

permits injection volumes of up to 5 pl isooctane solution without
loss of resolution (Figure 3b). This results in limits of detection for
A of about 0.1 ng/,ul with FID and 0.01 ng/pl with ECD detection.
On-column injection with an automatic sampler is possible when
an uncoated 0.53 mm i.d. column (0.5 m) replaces the 0.32 mmi.d.
retention gap and allows the use of stainless steel syringe needles.

PI

JI

PB

P II

f T

Figure 3a and b

'

'
6

CII

9 10 M I N

I'

Figure 2
HRGC-FID chromatogram (column 2) of A
(10 ng/pI) and pyrethrum extract
(630 ng/pI). On-column
injection (0.5 pl) was
used.

50

VOL. 14, JANUARY 1991

JII

$
4

:f

n, Y'

23
--.

MIN

10 M I N

HRGC-FID chromatogram (oncolumn injection,

column 2) of
bioallethrin (A), the
individual pyrethrins
from HPLC isolation,
piperonyl butoxide
(PB), tetramethrin
(T), and cypermethrin (Cy). a: each
at 10 nglpl except JI
and Jll at 8 ng/pl,
0.4 pl injected.
b: each at 0.1 ng/pI
except JI and Jll at
0.08 ng/pI, 4 pl
injected.

s-

Journal of High Resolution Chromatography

Optimized GC of Pyrethrins and Pyrethroids

4 Conclusions
Gas chromatographic analysis of natural pyrethrins and of various
pyrethroids in pyrethrum extract and in insecticidal formulations
is accomplished by means of HRGC with on-column injection
using hydrogen at high carrier gas velocities under constant flow
or programmed pressure conditions, and with short thin-film
capillaries. Resulting elution temperatures of thermolabile pyrethrin I and I1 are kept well below 200C with a non-polar
stationary phase, below 210 "C with a more polar stationaIy
phase. This avoids thermal degradation or isomerization and
allows quantitation of ideal gas chromatographic signals. The use
of a retention gap allows injection volumes of 4 pl or more on
manual or automatic injection resulting in detection limits of
about 0.1 ngipl for FID and 0.01 ngipl for ECD detection.

Figure 4
HRGC-FID chromatogram
(on-column injection,
pressure programmed,
column 3) of S-bioallethrin
(A) and the individual
pyrethrins from HPLC isolation (each at 100 nglpl
except JI and Jll at
80 ng/pl, 0.6 pl injected).

Thus insecticidal spray and powder formulations can be analyzed

conveniently for pyrethrins, pyrethroids, and synergist.
Acknowledgments
I

10 12

>
14 M I N

The continued support of Professor Dr. Karlheinz Ballschrniter is gratefully

acknowledged. Dr. habil. Manfred Mohnkeprovided helpful suggestioris on
the use of retention gaps.

References
[l] J.E.Casida, Environmental Health Perspectives 34 (1980) 189.

PI

[2] D.A Otieno, I.J. Jondiko, P.G McDowell. and F.J. Kezdy, J. Chrom.
Sci. 20 (1982: 566.
[3] R.J. Bushway. J. Assoc. Off. Anal. Chem.. 68 (1985) 1134
[41 A.M. McEldowney and R.C. Menary, J. Chromatogr. 447 (1988) 239.
[51 R.L. Holmstead and D.M. Soderlund, J. Assoc. Off. Anal. Chem 60
(1977) 685.

(61 K.R.Hill. Pure Appl. Chem. 51 (1979) 1617

171 Yoshihiko Kawano, Karl Yanagihara, Toru Miyamoto, and Izuru
Yamamoto. J. Chromatogr. 198 (1980) 317.

CI

181 L . Seda, G. Toninelli, and B. Sartorel, Riv. Ital. Sostance Grasse 60

(1983) 133.

PII

[91 A.F. Groneman, M.A Posthumus, L.G.M. Tuinstra, and W.A. Traag.
Anal. Chim. Acta 163 (1984) 43.

A J

[lo] N.S. Birdie, R.K.Banerij, and A K.Chauhan, Pyrethrum Post 16 (1985)

77.

Figure 5
HRGC-FID chromatogram (oncolumn injection,
column 2) of an
ethyl acetate extract (0.5 pI of 2 ml)
from spray no. 1.

[ll] R.W.
Stringham and R.P. Schulz, J. Assoc. Off. Anal. Chem. 68 (1985)

1137.
1121 W Ebing, Fresenius 2 . Anal. Chem. 321 (1985) 539.

1131 W. Dicke, H -D Ocker, and H -P. Thier. 2. Lebensm. Unters. Forsch.

186 (1988) 125.

Ib M I N -

I141 R C. Wieboldt, K.D. Kempfert, D.W.Later, and E.R. Campbell, HRC &
CC 12 (1989) 106.
1151 M. Elliot, J. Chem. SOC., Perkin I(1975) 1500

piperonyl butoxide (0.5 mgiml) (Figure 5) Spray no. 3 contains

higher concentrations of pyrethrum extract (1 mg/ml) and synergist (3 mgiml); additionally the pyrethroids tetramethrin and
cyphenothrin are present at concentrations of 0.5 and 1 mg/ml,
respectively. Though indicated on the label, spray no. 4 contains
no pyrethrum; tetramethrin, cypermethrin, and piperonyl
1,and 1.5 mg/ml,
butoxide are detected at concentrations of 0.3,O.
respectively. An insect powder no. 5 contains pyrethrum
(2.5 mg/g) and synergist (10 rng/ml).
Journal of High Resolution Chromatography

I161 T. Ando, Y . Kurotsu. and M. Uchiyana, Agric. Biol. Chem. 50 (1986)

491.
1171 T . J . Class, T. Ando, and J.E. Casida, J . Agric. Food Chem. 38 (1990)
529.
I181 G.Pattenden. L. Crombie, and P Hemesley, Org. Mass Spec. 7 (1973)
719.

Ms received: September 3, 1990

Accepted: December 14, 1990
VOL. 14, JANUARY 1991

51