Prohexadione-calcium

Materials to be

analyzed

Rice (grain, straw), wheat (grain), barley (grain), straw-

berry and soil

Instrumentation

High-performance liquid chromatographic determination

1

Introduction

Chemical name

(IUPAC)

Calcium 3-oxido-5-oxo-4-propionylcyclohex-3-ene-

carboxylate

Structural formula

O

O

O

O

O

2−

Ca

2

+

Empirical formula

C

10

H

10

CaO

5

Molar mass

250.3

Melting point

>360

◦

C

Vapor pressure

0.0133 mPa at 20

◦

C

Solubility

Water 174 mg L

−1

(20

◦

C), methanol 1.11 mg L

−1

(20

◦

C),

acetone 0.038 mg L

−1

(20

◦

C).

Stability

In water; DT

50

5 days at pH 5 and 83 days at pH 9

(20

◦

C). Stable to heat (200

◦

C). Under sunlight in water,

DT

50

4 days

Use pattern

Prohexadione-calcium, a plant growth regulator and

retardant, is used as an anti-lodging agent in small

grain cereals and it could also be used as a growth

retardant in turf, peanuts, flowers and to inhibit new

twig elongation of fruit trees.

Regulatory position

The residue definition is for the parent, prohexadione-

calcium, only.

2

Outline of method

Prohexadione-calcium in the samples is extracted with acidic acetone by shaking
(extracted as the free acid, prohexadione). The extract is purified by a series of

Handbook of Residue Analytical Methods for Agrochemicals.

C

2003 John Wiley & Sons Ltd.

Prohexadione-calcium

533

procedures of liquid–liquid partition, ion-exchange column chromatography, methyl
esterification (production of methyl ester of prohexadione) and reversed-phase col-
umn chromatography depending on the interfering materials in the analytical samples.
Prohexadione-calcium is determined by high-performance liquid chromatography
(HPCL) with ultraviolet (UV) detection (274 nm).

3

Apparatus

Mill (coffee-mill type)
Grinder (cutting mills, Willey type)
Blender (kitchen type)
Round-bottom flasks, 500-, 300-, 200- and 100-mL
Separatory funnels, 200- and 50-mL
Stoppered test-tube, 30-mL
Glass funnels, 10- and 4.5-cm i.d.
Condenser
Glass chromatography column (reversed-phase silica gel, 1.5-cm i.d.

× 40 cm, DEAE

ion exchanger, 1.0-cm i.d.

× 30 cm)

Reversed-phase silica gel column: Place a cotton wool plug at the bottom of a glass

chromatography column. Pack 5 g of reversed-phase silica gel slurried with a

solvent mixture of n-hexane–benzene–methanol (80 : 20 : 0.4, v/v/v) into the glass
column. Place an anhydrous sodium sulfate layer about 1-cm thick above and below

the silica gel bed

Bell jar-type filtering apparatus
Buchner funnel, 11-cm i.d.
Rotary vacuum evaporator, 40

◦

C bath temperature

Dry-block bath, electrically heated, temperature 75

◦

C

Mechanical shaker (universal shaker)
Ultrasonic cleaner
High-performance liquid chromatograph equipped with a UV detector
Microsyringe, 25-µL

4

Reagents

Acetone, acetonitrile, benzene, dichloromethane, n-hexane and methanol, pesticide

residue analysis grade

Chloroform, sodium chloride, anhydrous sodium sulfate, sulfuric acid (97%),

hydrochloric acid (36%), sodium bicarbonate, trifluoroacetic acid, tris(hydro-
xymethyl)aminomethane (Tris), special grade

Water, high-performance liquid chromatography grade
0.1 M Phosphate buffer solution (pH 7.0)
Reversed-phase silica gel, silica gel ODS-Q3, 75A, 30–50-µm (Wako Pure Chemical

Industries, Ltd)

DEAE ion exchanger, Cellulofine A-200 (Wako Pure Chemical Industries, Ltd)
Filter paper, 11-cm i.d.
pH test paper

534

Individual compounds

Prohexadione-calcium, analytical grade (Ihara Chemical Industries Co., Ltd)
Prohexadione-calcium standard solutions: 0.05, 0.2, 0.4, 0.6 and 0.8 mg L

−1

in ace-

tonitrile

Preparation of Tris–HCl buffer: Dissolve 60.5 g of Tris in 400 mL of distilled water,

add concentrated hydrochloric acid to adjust to pH 7.7 and add distilled water
to make exactly 500 mL to prepare a 1.0 M Tris–HCl buffer, which should be
stored at 5

◦

C. Prepare 0.05 M and 0.01 M buffers by diluting with distilled water

before use.

Preparation of ion-exchange column: Preparation of ion-exchange resin. To 500 mL

of Cellulofine 200A (DEAE ion exchanger), add 500 mL of water, mix well and
filter this mixture under reduced pressure. Wash the residue (ion-exchange resin)
twice with 300 mL of 0.5 M Tris–HCl buffer (pH 7.7) and filter this mixture under
reduced pressure. Swell the ion-exchange resin in 100 mL of the buffer for 1 h
to activate the resin. Remove the buffer by filtration and swell the ion-exchange
resin in 500 mL of 0.01 M Tris–HCl buffer (pH 7.7) for about 10 min. Remove the
buffer by filtration, swell and wash the ion-exchange resin three times with 200 mL
of the buffer for about 10 min each, and filter this mixture by suction. Swell the
washed ion-exchange resin in 100 mL of 0.01 M Tris–HCl buffer (pH 7.7) and
store as it is. Preparation of column. Plug the bottom of a glass chromatography
column of 1 cm i.d. with absorbent cotton and pack the column with 5 mL of the
prepared ion-exchange resin suspended in 0.01 M Tris–HCl buffer (pH 7.7) by
the wet method. Wash the resin in the column with about 20 mL of the buffer
before use.

Prohexadione-calcium standard solutions: Dissolve 10 mg of prohexadione-calcium

in 100 mL of water to prepare a 100 mg L

−1

solution. Transfer 100 µL of this

solution into a 30-mL test-tube, evaporate water to dryness under reduced pressure
and to methylate prohexadione-calcium according to Section 6.3. Dissolve the
product in acetonitrile to prepare the 0.05, 0.2, 0.4, 0.6 and 0.8 mg L

−1

acetonitrile

solutions.

5

Sampling and sample preparation

Collect 1 kg each of rice grain, wheat grain and barley grain and grind them with a mill.
Collect 1 kg of rice straw and grind it with a grinder. Collect 1 kg of strawberry and
homogenize with a blender. Collect soil, from the top10-cm surface layer, homogenize
and pass through a 5-mm sieve.

6

Procedure

6.1

Extraction

Weigh the samples [strawberry, rice grain, 25 g; wheat grain, barley grain, 10 g; rice
straw, 5 g; soil (dry weight basis), 30 g] in round-bottom flasks of appropriate volumes
(500- or 300-mL). For soil samples add 40 mL of 1 N sulfuric acid and 120 mL of
acetone to the flask, and for other samples add 20 mL of 1 N sulfuric acid and 60 mL

Prohexadione-calcium

535

of acetone to the flask. Shake the flask at room temperature for 30 min (prohexadione-
calcium is extracted as the free acid, prohexadione). Filter the extract through a filter
paper on a Buchner funnel with suction into a flask of an appropriate volume (500- or
300-mL). Wash the residue and the flask with 80 mL of acetone, and filter them in
a similar manner. Combine the filtrates and concentrate under reduced pressure (to
about 50 mL for soil and to about 20 mL for others).

Transfer the concentrate of soil sample extract into a 200-mL separatory fun-

nel with a small volume of water. To the concentrate, add 1 mL of concentrated
sulfuric acid and partition twice with 50 mL of n-hexane. Discard the n-hexane
layer.

Transfer the concentrated crop sample extract (strawberries, rice grain, barley grain

and rice straw) into a 50-mL separatory funnel with a small volume of water. Extract
the solution three times with 10 mL of a chloroform–methanol (3 : 1, v/v). Dry the
chloroform–methanol layer with a small amount (about 8 g) of anhydrous sodium
sulfate on a glass funnel and transfer the dried solution to a 100-mL separatory
funnel.

For wheat grain, extract the concentrate three times with 10 mL of chloroform–

methanol (3 : 1, v/v). For the soil sample, extract the aqueous layer after washing with
n-hexane twice with 60 mL of chloroform–methanol (3 : 1, v/v). Dry the chloroform–
methanol layer with anhydrous sodium sulfate [for wheat grain, use a small amount
(about 8 g) of anhydrous sodium sulfate] and collect the dried solution in a 200-mL
round-bottom flask. Evaporate the solvent under reduced pressure and proceed to
ion-exchange column chromatography.

Extract the chloroform–methanol layer from the strawberry, rice grain, barley grain

and rice straw samples twice with 30 mL of 0.1 M phosphate buffer solution (pH 7.0).
Since an emulsion is formed, the first extraction should be conducted with very gentle
shaking. Centrifuge the extract at 2500 rpm for 10 min, when an emulsion is formed.
Discard the chloroform–methanol layer.

Combine the aqueous layers, add 3.5 mL of concentrated sulfuric acid and ex-

tract the solution twice with 60 mL of a mixture of chloroform and methanol. Dry
the chloroform–methanol layer with anhydrous sodium sulfate and collect the dried
solution in a 200-mL round-bottom flask. Evaporate the solvent under reduced
pressure.

6.2

Ion-exchange column chromatography

To the flasks for the crop and soil samples (Section 6.1), add 2 mL of 0.01 M Tris–
HCl buffer solution (pH 7.7) and 50 and 100 µL of 1 M Tris–HCl buffer solution for
wheat grain, barley grain and rice straw, and for soil, respectively. Adjust the pH to
about 7.7 (confirm the pH with a pH test paper using the sample of untreated area).
Homogenize the residue with ultrasonication and transfer the homogenate to the top
of an ion-exchange column. Wash the flask twice with 2 mL of 0.01 M Tris–HCl
buffer solution and transfer the washings to the column. Elute the column with 40 mL
of the same buffer solution. Discard this eluate.

Subsequently, elute the target substance with 50 mL of the same buffer solution

containing 0.1 M sodium chloride. Transfer this eluate to a 200-mL separatory funnel,

536

Individual compounds

add 1 mL of concentrated sulfuric acid to the solution and extract twice with 50 mL
of chloroform–methanol (3 : 1, v/v). Dry the chloroform–methanol layer with anhy-
drous sodium sulfate and collect the dried solution in a 200-mL round-bottom flask.
Evaporate the solvent to dryness under reduced pressure.

6.3

Methylation

Transfer crop and soil samples from Section 6.1 (strawberry and rice grain) and
Section 6.2 with 5 mL of methanol to 30-mL test-tubes and add to each test-tube
0.05 mL of concentrated sulfuric acid. Attach a condenser and reflux the solution at
75

◦

C for 60 min to esterify prohexadione to its corresponding methyl ester. Cool the

reaction mixture to room temperature, add 20 mL of water and extract the reaction
solution twice with 20 mL of dichloromethane. Dry the dichloromethane layer with a
small amount of anhydrous sodium sulfate and collect the dried solution in a 100-mL
round-bottom flask. Evaporate the solvent under reduced pressure.

For soil samples, dissolve the residue in an appropriate volume of acetonitrile prior

to HPLC analysis.

Dissolve the crop residue samples from above in 0.5 mL of dichloromethane and

add 20 mL of n-hexane to the solution. Transfer the mixture to a 50-mL separatory
funnel, add 20 mL of 0.2 M sodium bicarbonate solution and shake the funnel. Since
an emulsion may be formed during shaking, initially shake the funnel very gently.
Centrifuge at 2500 rpm for 10 min, if necessary. Collect the aqueous layer and discard
the n-hexane layer.

Add 0.8 mL of concentrated sulfuric acid (pH 2–3) to the aqueous layer and extract

twice with 20 mL of dichloromethane. Dry the dichloromethane layer with anhy-
drous sodium sulfate and collect the dried solution in a 100-mL round-bottom flask.
Evaporate the solvent under reduced pressure.

For wheat grain, dissolve the residue in an appropriate volume of acetonitrile prior

to HPLC analysis.

6.4

Reversed-phased silica gel column chromatography cleanup

Dissolve the crop residue samples from Section 6.3 in 0.5 mL of dichloromethane.
Transfer this solution to the top of the reversed-phased silica gel column with
4.5 mL of n-hexane–benzene–methanol (80 : 20 : 0.4, v/v/v) and elute with the same
solvent. Discard the first 70-mL of the eluate and collect the second 100-mL
of eluate in a 200-mL round-bottom flask. Evaporate the solvent under reduced
pressure.

Dissolve the residue in an appropriate volume of acetonitrile for HPLC analysis.

6.5

High-performance liquid chromatographic determination

Inject an aliquot (V

i

) of the soil and crop samples into the HPLC system.

Prohexadione-calcium

537

Operating conditions
Instrument

LC-10AD equipped with an SPD-10A UV spectrophoto-
metric detector (Shimadzu Co., Ltd, Japan)

Column

CAPCELL PAK C

18

SG 120 (Shiseido Co., Ltd, Japan),

4.6-mm i.d.

× 250 mm; column temperature, ambient

Mobile phase

Acetonitrile–distilled water–trifluoroacetic acid
(20 : 30 : 0.1, v/v/v)

Flow rate

1.0 mL min

−1

Wave length

274 nm

Attenuation

0.002 absorbance

Chart speed

10 mm min

−1

Injection volume

1–10 µL

Retention time

8.4 min

Minimum detectable

amount

0.5 ng

7

Evaluation

7.1

Method

Quantitation is performed by the calibration technique. Construct a fresh calibration
curve with prohexadione-calcium standard solutions for each set of analyses. Inject
10 µL of each prohexadione-calcium standard solution into the HPLC system. Using
log–log paper, plot the peak heights in millimeters against the injected amount of
prohexadione calcium in nanograms. Also inject 1–10-µL aliquots of the sample
solutions. For the heights of the peaks obtained for these solutions, read the appropriate
amounts of prohexadione-calcium from the calibration curve.

7.2

Recoveries and limits of detection

The recoveries from control samples fortified with prohexadione-calcium at a level of
0.2 mg kg

−1

were 75–80% for strawberry, 79–83% for rice grain, 79–89% for wheat

grain, 92–105% for barley grain and 81–89% for soil. The recoveries from control
samples fortified with prohexadione-calcium at a level of 0.5 mg kg

−1

were 69–75%

for rice straw. The limits of detection were 0.01 mg kg

−1

for wheat grain and soil,

0.02 mg kg

−1

for strawberry, rice grain and barley grain and 0.05 mg kg

−1

for rice

straw.

7.3

Calculation of residues

The residue R, expressed in mg kg

−1

prohexadione-calcium, is calculated from the

following equation:

R

= (W

A

× V

End

)

/(V

i

× G)

where

538

Individual compounds

G

= sample weight (g)

V

End

= terminal volume of sample solution (mL)

V

i

= portion of volume V

End

injected into HPLC system (µL)

W

A

= amount of prohexadione calcium for V

i

read from calibration curve (ng)

8

Important points

1. Since prohexadione-calcium degrades rapidly in soil, soil samples should be ana-

lyzed or frozen immediately after sampling.

1

2. The extraction method for prohexadione-calcium in soil was developed using allu-

vial soil and volcanic ash soil. Extraction by shaking the soil with a mixture of 1 N
sulfuric acid–acetonitrile (1 : 3, v/v) and/or of 1 N sulfuric acid–acetone (1 : 3, v/v)
showed an acceptable extraction recovery efficiency.
Prohexadione in aqueous solution is not partitioned in n-hexane. More than 85%
of prohexadione in solutions of pH 4 or lower is partitioned in ethyl acetate.

3. Methyl esterification: When prohexadione was treated with methanolic

HCl (3%, w/v) or sulfuric acid–methanol (1%, v/v) under reflux at 75

◦

C for 60 min,

or with BF

3

–methanol (14%, w/v) under reflux at 75

◦

C for 30 min, the yield of

the methyl ester of prohexadione was 95, 93 and 82%, respectively (prohexadione,
10 µg, volume 2 mL of methanolic HCl, 2 mL of sulfuric acid–methanol and 1 mL
of BF

3

–methanol). A solution of 1% (v/v) sulfuric acid in methanol was chosen

for ease of preparation. Even if prohexadione was treated with 1% sulfuric acid
in methanol at room temperature for 12 h, the yield of prohexadione methyl ester
was not different from that under reflux conditions as described in Section 6.3. The
conditions for methyl esterification in Section 6.3 were chosen because of short-
ening of the analysis time and the reproducibility of the reaction yield in residue
analysis samples which could contain large quantities of contaminants.
Since the methyl ester of prohexadione had lower polarity compared with prohexa-
dione, an ODS column was very useful for purifying the sample.
Since the methyl ester of prohexadione degrades rapidly in 1 M NaOH, it should
not be handled under alkaline conditions.

4. Prohexadione-calcium degrades in aqueous sodium hypochlorite solution.

Prohexadione-calcium at the level of 0.08 mg kg

−1

in tap water degrades and dis-

appears rapidly.

2

Degradation of prohexadione-calcium can be prevented by addi-

tion of ascorbic acid at about 1 mg kg

−1

in tap water. Degradation products of pro-

hexadione-calcium by aqueous chlorination are identified by mass spectrometry.

5. Sample storage stability: Prohexadione-calcium in strawberry, rice grain, rice

straw, wheat grain and barley grain is stable at

−20

◦

C for 40, 140, 60, 80 and

100 days, respectively. Approximately 88% of the applied prohexadione-calcium
remained in soil when stored at

−20

◦

C after 80 days.

References

1. A. Yagi, ‘An example of the evaluation of the behavior in the environment of new agricultural

chemicals,’ in “Proceedings of the 12th Symposium on Environmental Science of Pesticides,”
pp. 19–25 (1994) (in Japanese).

Prohexadione-calcium

539

2. M. Ikeda, Y. Asano, and Y. Yusa, ‘Degradation of prohexadione-calcium by aqueous chlorination,’

in “Abstracts of the 20th Annual Meeting of the Pesticide Science Society of Japan,” p. 152
(1995).

3. A. Yagi, K. Mizuno, Y. Asano, and K. Ishikawa, ‘Residue analytical method for the calcium

salt of 3,5-dioxo-4-propionylcyclohexane-1-carboxylic acid (prohexadione-calcium) in crops,’
in “Abstracts of the 16th Annual Meeting of the Pesticide Science Society of Japan,” p. 114
(1991).

Akira Yagi, Mitsumasa Ikeda and Yoshihiro Saito

Kumiai Chemical Industry Co. Ltd, Shizuoka, Japan