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INTRODUCTION

Research associated with the development of new crop protection

chemicals centers around the design of products that provide highly

effective and specific action towards the target organism with

reduced application rates. It is estimated that 25-30% of the

pesticides on the market today have optical isomers. The desired

activities often result from a single enantiomer in the optical isomer

mixtures. It is therefore important to assess the enantiomeric purity

of the chiral active ingredients in the formulation. In addition, the

detection, characterisation and quantitation of the other components

in the formulation are necessary to support product registration.1

There has been an increasing adoption of supercritical fluid

chromatography (SFC) on chiral stationary phases for chiral

separation. The properties of a supercritical fluid, its high diffusivity

and low viscosity in particular, enable high efficiency chiral

separations with shorter run times. UltraPerformance Convergence

Chromatography (UPC2) applies the performance advantages of

UPLC® to SFC, combining the use of supercritical CO2 with sub-2-μm

particle columns. UPC2 is an orthogonal analytical technique to

reversed-phase LC and can be used to solve complex separations

challenges.

In this study the achiral screen of a formulated pesticide product

using UPC2 showed that the minor components detected using UV

and mass detection had similar structural characteristics to the

active ingredient (AI), propiconazole, a triazole fungicide with two

chiral centers (Figure 2). The minor components had the same m/z

and shared the same isotopic pattern as the AI. Subsequent chiral

resolution of the propiconazole in the formulation in combination

with simultaneous UV and mass detection provided valuable spectral

information which allowed the minor components to be characterised

as probable stereoisomers.1

Chiral and Achiral Profiling of a Pesticide Formulation

using UltraPerformance Convergence Chromatography

(UPC2) with PDA and Mass Detection

Marian Twohig,1 Jane Cooper,2 Hélène Boiteux,3 John McCauley4 1Waters Corporation, Milford, MA, USA; 2Waters Corporation, Wilmslow, UK; 3Waters Corporation, 5 rue Jacques Monod, 78280 Guyancourt, France. 4Waters Corporation, New Castle, DE, USA.

PDA

QDa

Figure 1. Waters® ACQUITY QDa Detector is a novel mass

detector that can be integrated into existing liquid chromatography configurations in order to increase sensitivity

and complement the results obtained using UV detectors.

Table 1. Summary of the analysis conditions.

RESULTS AND DISCUSSION Achiral Separation

ACQUITY UPC2 chromatograms (Figure 2) of the

propiconazole standard (lower trace) and the formulation

sample (top trace) obtained using an ACQUITY UPC2 BEH

column.

The retention times of peaks 1 and 2 (propiconazole

diastereomers) in the formulation sample matched those of

the propiconazole standard.

Two minor peaks (peaks 3 and 4) were observed in the

formulation sample with retention times (tR) of 2.22 min and

tR 2.26 min, respectively.

The UV spectra of peaks 3 and 4 resemble those for peaks 1

and 2 (Figure 2), indicating their structural similarity. In

addition, the four peaks resulted in identical mass spectra

with base peaks at m/z 342 and an isotopic pattern

characteristic of dichlorinated compounds. The m/z matched

the protonated propiconazole.

Figure 2. ACQUITY UPC2 UV achiral separation of the propiconazole

present in the formulation sample and propiconazole standard at 220 nm using the ACQUITY UPC2 BEH Column. The UV and MS spectra for peaks 1-

4 are also shown.

Chiral Separation

The ACQUITY UPC2 System has multi-column switching capabilities and a choice of up to four co-solvents which

allows both achiral and chiral method development and sample analysis to be performed on the same system.

Using gradient separation on an ACQUITY UPC2 Trefoil AMY1

chiral column (Figure 3). The two diastereomer peaks of propiconazole observed in Figure 2 were baseline resolved

into four individual peaks (1-4). Interestingly, the two minor isomer peaks in Figure 2 were also resolved into four peaks

(5-8) in a comparable manner, indicating a similar chirality for propiconazole and the minor components.

References

1. Twohig, M. O’Leary, M. McCauley, J.P. Chen, R. Chiral and Achiral Profiling of

a Pesticide Formulation Using the ACQUITY UPC2 System and the ACQUITY

QDa Detector. Waters application library. APNT134790013.

2. Glaser, R. Adin, I. Ovadia, D. Mendler, E. Drouin, M. (1995) Solid-state

structure determination and solution-state NMR characterization of the

(2R,4R)/2S,4S)- and (2R,4S)/(2S,4R)-diastereomers of the agricultural

fungicide propiconazole, th(2R,4S)/(2S,4R)-symmetrical triazole

constituational isomer, and a ditriazole analogue. Structural Chemistry, Vol

6, No 3. 145-156.

3. McCauley, J.P. Twohig, M. Chen, Rui. Isolating Trace Impurities for

Structural Elucidation in a Commercial Fungicide Formulation Using

Preparative SFC. Chirality 2014 Prague, Czech Republic. July 27th to 30th

2014. Waters application library PSTR134807862.

CONCLUSION The addition of mass detection as a complementary

analytical detection technique enhances confidence

in compound detection and identification.

The ACQUITY UPC2 System has column switching

capabilities so that both chiral and achiral columns

can be used with a choice of four co-solvents that are

compatible with MS analysis. The chiral and achiral

method development and analysis can be performed

on the same system.

The ACQUITY UPC2 System allows high efficiency

separations that can increase sample throughput

compared to traditional normal-phase separations.

The diasteromeric resolution of propiconazole using

UPC2 took place in less than 3 minutes, which is at

least 10 times faster than normal phase methods

reviewed in the literature.1

Matrix components visible in the UV chromatogram and the MS total ion chromatogram (TIC) of the formulation

sample are clearly differentiated from the isomeric peaks of interest using an extracted ion chromatogram (XIC)

(inset Figure 3).

The detection sensitivity and selectivity of the method are improved when using mass detection in combination with

UV detection which is used in order to measure the enantiomeric purity of the chiral pesticide in the

formulation.

Based on the observations, it is postulated that the minor component is a regioisomer of propiconazole. A

regioisomer of propiconazole originating from one of the

nitrogens on the triazole ring was characterised by Glaser et al.2

Further experiments to characterise and isolate this

compound for positive identification were carried out. Methods were developed to scale separations to SFC

preparative chromatography, each minor stereoisomer was isolated followed by characterisation using 1H, 13C,

2D and NOE NMR spectroscopy. These experiments revealed that the minor isomer peaks differed from

propiconazole itself by the the triazole nitrogen attachment point to the methylene group that is bridging

to the dioxolane ring. The assigned structures for the all of the isolated components were in agreement with the

information in the prior literature by Glaser et al.2,3

Figure 3. ACQUITY UPC2 UV chromatogram at 220 nm showing the chiral resolution of the propiconazole stereoisomers and unknown

chiral components in the formulation sample using the ACQUITY UPC2 Trefoil AMY1 column. The XIC of m/z 342 and the UV and MS spectra for each peak are also shown.

Reproducibility

Empower 3 can be used to assess the peak attributes with its

system suitability function. Chromatographic parameters can

be determined and statistical measurements can be made on

them. In this example the RSDs for 10 determinations of the

retention time, area, area % height and USP resolution were

< 0.61% for all of the propiconazole stereoisomers (Table 2).

Table 2. RSD for ten replicate injections of the propiconazole stereoisomers in the formulation sample.

%RSD (n=10)

PropiconazoleStereoisomers

tR Area Area % Height USP Resolution

Peak 1 0.37 0.44 0.09 0.61

Peak 2 0.33 0.39 0.05 0.52 0.43

Peak 3 0.27 0.37 0.05 0.57 0.29

Peak 4 0.22 0.37 0.04 0.61 0.21

342

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m/z

320.00 325.00 330.00 335.00 340.00 345.00 350.00 355.00 360.00

256.9 269.9 307.9 341.0

AU

0.000

0.001

0.002

271.1 337.6

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0.001

0.002

219.2

269.9 307.9 327.0 338.8

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0.003

266.4 319.8 352.7

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PDA 220 nm

XIC m/z 342

12

3 4

58

6 7

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XIC m/z 342

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0.00 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 3.00 3.20 3.40 3.60 3.80 4.00 4.20 4.40 4.60 4.80 5.00 5.20 5.40 5.60 5.80 6.00 6.20

1 2

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4.60 4.80 5.00 5.20 5.40 5.60 5.80 6.00

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-0.010

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-0.010

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4.60 4.80 5.00 5.20 5.40 5.60 5.80 6.00

PDA 220 nm

5 6 7 8

XIC m/z 342

5 6 7 8

PDA 220 nm

QDa XIC m/z 342

Rs1,2 = 2.42Rs2,3 = 2.28Rs3,4 = 2.40

METHODS Achiral Gradient Conditions:

0 min 3% B, 4 min 30% B, 6 min 30% B, return to initial

conditions.

Chiral Gradient Conditions:

0 min 7% B, 3 min 11% B, 6 min 17% B, 7 min 40% B, return to

initial conditions.

Mass Detector Conditions

MS system: ACQUITY QDa Detector

Ionization mode: ESI+ ; Capillary voltage: 1.0 kV

Desolvation temp.: 600 °C ; Source temp.: 150 °C

Cone voltage: 10 V; Sampling rate: 5 Hz

MS scan range: 100 to 600 m/z

Empower 3 FR2 Software was used for data acquisition and

chromatographic data processing.

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2.20 2.40

12

12

Formulation

sample

Propiconazole standard

3

4

1

2

3

4

1

2

3

4

34

(2R,4R)-propiconazole

(2R,4S)-propiconazole

(2S,4S)-propiconazole

(2S,4R)-propiconazole

UV SpectraPDA 220 nm MS Spectra

Achiral Chiral

Separation mode Gradient Gradient

Column ACQUITY UPC2 BEH, 3.0 x 100 mm, 1.7-μm

ACQUITY UPC2 Trefoil AMY1 3.0 x 150 mm, 2.5-µm

Co-solvent (B) Methanol 50:502-propanol/ethanol

ABPR 1990 psi/137 bar 3000 psi/207 bar

Flow rate 1.5 mL/min 1.5 mL/min

UV detection 220 nm 220 nm

Column temp. 35 °C 20 °C

Injection volume 0.5 µL 0.5 µL

Make-up solvent 98:2 methanol:water with 0.1% ammonium hydroxide at 0.3 mL/min