Direct Chiral Separation of Pyrethroid Isomers by HPLC with Chiral

Chapter 3

Direct Chiral Separation of Pyrethroid Isomers by HPLC with Chiral Stationary Phases

Downloaded by CORNELL UNIV on July 26, 2012 | http://pubs.acs.org Publication Date (Web): July 25, 2012 | doi: 10.1021/bk-2012-1099.ch003

Masahiko Okamoto* Organic Synthesis Research Laboratory, Sumitomo Chemical Co. Ltd., 3-1-98 Kasugade-naka, Konohana-ku, Osaka 554-0022, Japan *E-mail: [email protected]

Pyrethroids are synthetic pesticides with a chemical structure based on natural pyrethrins to enhance photostability and insecticidal activity. They are the family of chiral pesticides with a large number of stereoisomers. Enantiomers of pyrethroids have different biological activity and toxicity against mammals so the development of reliable chiral analytical methods for the determination of individual stereoisomers is indispensable. Several techniques have been developed for this purpose including gas chromatography, high performance liquid chromatography (HPLC), and so forth. In this chapter, the analysis of synthetic pyrethroids by HPLC with chiral stationary phases including some historical background of this line of research is described.

Introduction The agrochemical industry has continuously been probing for new active compounds with lower application rates, increased selectivity and decreased undesired ecological impact to control pests. Many agrochemicals including pyrethroids are chiral and each stereoisomer may have different properties and effectiveness. When two stereoisomers are mirror images of each other, they form an enantiomer pair. Each stereoisomer has only one enantiomer. Enantiomers are chemically identical, having the same boiling points, melting points, solubility, © 2012 American Chemical Society In Parameters for Pesticide QSAR and PBPK/PD Models for Human Risk Assessment; Knaak, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.

reactivity, and other chemical properties. A detailed analysis of the correlation between biological activity and stereochemistry of the active principles shows clearly that the situation is far from being black and white (1). Typical situations frequently met are the following: 1. 2. 3.


4.

The stereoisomers have complementary biological activity All stereoisomers possess nearly identical quantitative and qualitative biological activity The stereoisomers have qualitatively similar activities but quantitatively different potencies. The stereoisomers have qualitatively different biological activities (the desired biological activity residues in one stereoisomer).

The occurrence of racemates and single isomers among pesticides is shown in Figure 1. A rough statistical analysis of the entries of the Pesticide Manual 15th Edition shows that products containing stereoisomerically enriched active ingredients are established technology: approximately 23% of all entries (carbon)chiral compounds (2). 8% of the listed compounds are commercialized as single or enriched isomers.

Figure 1. The occurrence of racemates and single isomers among pesticides on the market (2). *Includes biological pesticides which fungi or bacteria are used as active ingredients.

One of the most significant differences of pyrethroids in comparison with many other pesticides is that most of them have one to three chiral centers. Usually, commercial formulations are sold as several isomers of one pyrethroids. These compounds present enantiomeric selectivity, with biological activity 32 In Parameters for Pesticide QSAR and PBPK/PD Models for Human Risk Assessment; Knaak, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.

generally residing in only one of the enantiomers. This enantiomeric selectivity has important implications in the manufacture and use of chiral agrochemicals in general.


Structure-Activity Relationship of Pyrethroids Pyrethroids are synthetic pesticides with a chemical structure based on pyrethrins. The basic pyrethrin structure was altered such that the developed products, the pyrethroids, have enhanced photostability, insecticidal activity, and also mammalian toxicity (3). Pyrethroids are composed of two basic structural moieties, an acid and an alcohol. An important aspect of the chemical and toxicological properties of pyrethroids is their overall configuration. A pyrethroid’s configuration influences its toxic potency and predominant pathway for metabolism. As shown in Figure2, the cis and trans designation indicates how a substituent on carbon-3 of the cyclopropane ring is oriented in relation to the carboxylic acid group bound to carbon-1. Cis implies the carboxylic acid on carbon-1 and the substituent on carbon-3 are oriented on the same side, whereas trans implies they are on opposite sides.

Figure 2. Stereochemical configurations of the chrysanthemic acid moieties. Table 1 shows that efficacy of prallethrin isomers against houseflies (4). Among 8 stereoisomers, the most insecticidal compounds have the structure of 1R trans followed by the 1R cis configuration. Those that have the structure of 1S cis or 1S trans are the least active.On the other hand, the most toxicologically active 33 In Parameters for Pesticide QSAR and PBPK/PD Models for Human Risk Assessment; Knaak, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.

compounds in mammals have the structure of 1R cis followed by the 1R trans configuration. Those that have the structure of 1S cis or 1S trans are the least active (5). Therefore, for a full understanding of structure-activity relationship of pyrethroids, it is essential to be able to monitor individual stereoisomers.

Table 1. Efficacy of prallethrin isomers against Houseflies isomer


alcohol portion

acid portion

LD50 (μg/♀

S

(1R)-trans

0.043

S

(1R)-cis

0.11

R

(1R)-trans

0.25

R

(1R)-cis

0.32

S

(1S)-trans

3.30

S

(1S)-cis

10.8

R

(1S)-trans

18.5

R

(1S)-cis

28.2

Reference: natural pyrethrin

0.73

Chiral Separation Methodology The chiral separation methodologies for the determination of optical isomers are mainly categorized into GC and HPLC. HPLC is a more useful technique for the separation of enantiomer pairs than GC, because it is quite rapid, nondestructive and there is very little possibility of epimerization during the analysis as observed in GC. Chiral separation with chromatography can be classified as two methods, indirect and direct method. Indirect method is the chromatographic methodology which separates the diastereomeric isomers after derivatization of optically pure reagent (6). This implies that separation of enantiomers can not be directly performed chromatographically. It is based on the concept that the enantiomers can be made separable on achiral column by appropriate derivatization to form diastereomers, because diastereomers have different physical and chemical properties. 34 In Parameters for Pesticide QSAR and PBPK/PD Models for Human Risk Assessment; Knaak, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


On the other hand, in the case of direct method, so-called “chiral chromatography” chiral stationary phase (CSP) is used. It is usually an optically active compound, causing a difference in retention between the enantiomers. In the case of indirect method, if the optically pure reagent which is used for derivatization is not 100% optical pure, precise optical purity of enantiomer will not be obtained. Additionally, it needs a complicate operation such as hydrolysis and derivatization (6). Therefore, the direct method is considered as being more preferable than indirect method. There are over 100 HPLC chiral columns on the market since the first commercially available HPLC chiral column was introduced by Pirkle and Fenn in 1981 (7). The large number of CSPs presents the analysis with several different possibilities in the development of an assay, but also raises the question of which CSP to choose. It involves the classification of CSPs based on how the solute-CSP complexes are formed. In chiral HPLC the selectors used in the CSPs include some low-molecular-weight molecules such as Pirkle’s type compounds, polysaccharides, cyclodextrins, polyacrylamides, proteins, macrocyclic antibiotics, and so forth (8). Figure 3 shows the analyte-CSP interaction proposed as chiral recognition model interpreted by Prof. Pirkle, we call it Pirkle type CSP, which is mainly used for the enantioseparation of pyrethroid isomers (9). The great success of this technique was interpreted in the framework of Dalgliesh’s three-point interaction theory, as being a result of a combination of simultaneous π-π interaction and hydrogen bonding in the non-polar solvent used as the mobile phase (8).

Figure 3. Chiral recognition model interpreted by Prof. Pirkle. The charge-transfer complex formation causes simultaneous additional enantiomer-dependent contacts between the partners. (Reproduced with permission from reference (8). Copyright 1990 John Wiley and Sons.) 35 In Parameters for Pesticide QSAR and PBPK/PD Models for Human Risk Assessment; Knaak, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.

Chiral Separation of Pyrethroids


In 1970s, the chiral columns with CSP were not commercially available. Thus, the chiral separations in the initial stage were performed using indirect method (10). Figure 4 shows the procedure to separate optical isomers of d-phenothrin. The 4 optical isomers of d-phenothrin were hydrolyzed in alkaline conditions to liberate chrysanthemic acid, which was derivatized to diastereomeric (+)-2octanol esters, and isomer ratio was determined by GC. It can be seen in Figure 4 that 4 isomers could be separated from one another.

Figure 4. Gas chromatogram of the separation of optical isomers of d-phenothrin on glass capillary column. (Reproduced with permission from reference (11). Copyright 1985 AOAC International.)

Prompted by the advent of Pirkle’s CSP, Sumitomo Chemical Co. Ltd. developed the Sumichiral OA type chiral HPLC columns which had Pirkle’s type CSPs (11) to separate the intact pyrethroids. A typical chromatogram of d-phenothrin optical isomers on Sumichiral OA2000 column ((R)-phenylglycine and 3,5-dinitrobenzoic acid) is shown in Figure 5. The complete separation of 4 isomers was achieved when using these columns in series (11). The data obtained by GC and chiral HPLC show good agreement for optical isomers of five insecticidal pyrethroids (Table 2) (12). These chiral HPLC methods have been used for quality control of these insecticidal pyrethroids as well as compendial analytical methods in Japanese Insecticides Directive since 1990. The Sumichiral OA-2000 column was also employed for the enantioseparation of other pyrethroids such as fenvalerate, cypermethrin and cyfluthrin (13). 36 In Parameters for Pesticide QSAR and PBPK/PD Models for Human Risk Assessment; Knaak, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


Figure 5. LC Chromatogram of the separation of d-phenothrin isomers on Sumichiral OA-2000 chiral columns in series.

Table 2. Comparison of the results from direct HPLC and indirect GC method (%; optical isomer ratio) Lot B

Lot A

Lot C

HPLC

GC

HPLC

GC

HPLC

GC

dl-d-T80-Allethrin

95.7

95.7

95.5

95.5

95.7

95.8

d-T80-Futharthrin

96.1

95.1

95.9

95.6

96.0

95.7

d-T80-Resmethrin

95.8

95.4

96.4

96.6

96.4

95.9

d-T80-Furamethrin

95.8

96.1

95.8

95.9

96.1

96.0

d-Phenothrin

95.6

96.0

96.9

96.1

95.6

95.5

HPLC method; Direct method (Sumichiral OA-2000 columns in series); GC method; Indirect method (Hydrolysis/diastereomer method, derivatization reagent); (+)-2-octanol, column: QF-1 (achiral column).

Besides Sumichiral OA-2000 chiral column, modified Pirkle’s type chiral columns, were efficient for the enantioseparation of ten pyrethroidal insecticides with three different columns (14): Sumichiral OA-4000 ((S)-valine and (S)-1-(α-naphthyl)-ethylamine), Sumichiral OA-4600 ((S)-tert-leucine and (S)-tert-leucine and (S)-1-(α-naphthyl)-ethylamine), OA-4700 (urea 37 In Parameters for Pesticide QSAR and PBPK/PD Models for Human Risk Assessment; Knaak, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


derivative derived from (R)-1-(α-naphthyl)-ethylamine with (S)-tert-leucine), and Sumichiral OA-2500 ((R)-1-naphthylglycine and 3,5-dinitrobenzoic acid). Each chiral column could separate the stereoisomers of pyrethroids with n-hexane-dichloroethane-ethanol in different proportions as mobile phases. For example, allethrin was partially resolved using a Sumichiral OA-2000 chiral column. However, an efficient separation of allethrin into eight isomers was accomplished with Sumichiral OA-4600 chiral column. The chromatogram of bioallethrin clearly shows that this compound consists of (αS)(1R) trans and (αR)(1R) trans diastereomeric pair. The separation of eight isomers of cypermethrin, which contain one chiral center in alcohol portion and two chiral centers in the acid portion, was difficult using Sumichiral OA-2000 chiral column. The separation with Sumichiral OA-2500, 4600, and 4700 chiral columns was also incomplete. However, the combination of two chiral columns, Sumichiral OA-4600 and 4700 gave a sufficient separation of eight isomers of cypermethrin (Figure 6). The good selectivity of Sumichiral OA-4700 for diastereomeric isomers may contribute to successful separation of eight isomers (14). Sumichiral OA-2500 column was also capable to separate the stereoisomers of cis-bifenthrin with n-hexane-1,2-dichloroethane (15, 16) or n-hexane-IPA (2-propanol)-ethanol (9980:6:14) as mobile phases (17). Additionally, Sumichiral OA-2000I and OA-2500I chiral columns whose CSPs consist of (R)-N-(3,5-dinitrobenzoyl)-1-naphthylglycine ionically bonded to γ-aminopropyl silanized silica which contains either a dinitrobenzoyl group or a naphthyl group attached to an asymmetric carbon, could separate the stereoisomers of fenpropathrin and terallethrin (18).

Concluding Remarks and Future Trends In every case, the results presented here, the extensive optimization of analytical conditions was conducted by changing the mobile phase composition, column temperature, or by combining two columns in series, and so forth. It should be emphasized that fine tunings of separation conditions are critical to establish reliable analytical methods. Even now, the chiral separation of pyrethroids is a challenging target, because pyrethroids can have one to three chiral centers. With the development of column technology, polysaccharides based CSPs have been introduced (19). These columns have applicability for a wide range of classes of compounds (20). In fact, these columns are promising for alternatives of enantioseparation of pyrethroids (21). Especially, for Type I pyrethroids (no cyano group) polymeric CSPs based on cellulose-derivatives seemed to be the most suitable for their resolution, while multiple interaction Pirkle’s type CSPs showed advantages in the enantiomeric separation of α-cyano pyrethroids (22).

38 In Parameters for Pesticide QSAR and PBPK/PD Models for Human Risk Assessment; Knaak, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


39 Figure 6. Separation of Cypermethrin isomers (a) with Sumichiral OA-4600; (b) with Sumichiral OA-4600 and OA-4700 in series. (Reproduced with permission from reference (14). Copyright 1990 Elsevier Limited.)

In Parameters for Pesticide QSAR and PBPK/PD Models for Human Risk Assessment; Knaak, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.

References 1. 2. 3.

4.


5. 6.

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9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19.

20. 21. 22.

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40 In Parameters for Pesticide QSAR and PBPK/PD Models for Human Risk Assessment; Knaak, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.

Direct Chiral Separation of Pyrethroid Isomers by HPLC with Chiral

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