Injection volume, mobile phase, mL water, Column temperature, phosphate buffer, Sample Preparation, ammonium phosphate, river water, LC, Kevin M. Jenkins Waters Corporation Oasis, SPE, Michael S. Young, cation-exchange, HPLC, Quaternary Ammonium Compounds
THE APPLICATION NOTEBOOK -- September 2004
Oasis® WCX: A Novel Mixed-Mode SPE Sorbent for LCMS Determination of Paraquat and Other Quaternary Ammonium Compounds Michael S. Young and Kevin M. Jenkins Waters Corporation
Oasis® WCX, a mixed-mode weak cation-exchange SPE cartridge, provides unique retention of paraquat and related compounds. An Atlantis® HILIC column was utilized for HPLC using no ion-pairing reagents. P araquat and diquat, quaternary ammonium compounds (quats), are high-use agricultural chemicals. The 1997 USGS estimated use of the herbicide paraquat was over 3,000,000 lb, and the estimated use of diquat was about 200,000 lb (see Figure 1). Figure 1: The structures of paraquat and diquat. Because these substances are ionic, solid-phase extraction (SPE) and HPLC usually have been accomplished with the aid of an ion-pairing reagent such as an alkyl sulphonic acid. liquid chromatography
(MS) provides the analyst with a more sensitive and highly selective analytical method for these compounds. Strong catIon Exchange
-based SPE methods (Oasis® MCX) that do not require ion-pairing reagents have been employed successfully for quats SPE using LCUV, but the strong salts or strong acid
s used for elution are difficult to remove and are serious impediments to optimal LCMS analysis. In order to overcome these problems, a new type of sorbent has been developed for the retention of quaternary ammonium compounds and strongly basic organic compounds
. The Oasis® WCX sorbent incorporates a weak cation-exchanger bound to a polymeric reversed-phase particle. Using the new sorbent and HILIC-based tandem LCMS, the quantitation limit for paraquat is below 100 ng/L (ppt) using only 20 mL of sample. Prior methods required larger sample volumes and were better suited for LCUV. experimental conditions
Instruments: Waters Alliance® 2695 separations module equipped with column heater and sample chiller, Waters/Micro-
mass Quattro MicroTM mass spectrometer, Waters 2487 dual wavelength UV detector. Sample Preparation A 20-mL river water
sample was adjusted to pH 7 by dropwise addition of 1 M ammonium phosphate
buffer. SPE Procedure 1. Condition a 60 mg, 3 cc Oasis® WCX SPE cartridge with 1 mL methanol and 1 mL water. 2. Load 20-mL river water sample. 3. Wash the cartridge with 1 mL of 10 mM pH 7 phosphate buffer, 1 mL water, and 1 mL of methanol. 4. Elute with 1.5 mL ACN/water/TFA 84:14:2. 5. Reconstitute in 0.5-mL mobile phase. LC Conditions Column: Atlantis® HILIC, 2.1 150 mm, 3.5 m Mobile phase: 40% acetonitrile, 60% aqueous buffer pH 3.7 (250 mM ammonium formate) flow rate
: 0.4 mL/min Injection volume: 20: L column temperature
: 30 °C Alternate column for LCUV: Atlantis dC18, 2.1 150, 3.5 m Mobile phase: 25:75 acetonitrile/0.1% heptafluorobutyric acid in Water flow
rate: 0.3 mL/min Injection volume: 20: L Column temperature: 30 °C Monitored MS Transitions
Table I: MS transitions for paraquat and diquat
MW MRM Cone Voltage Collision Energy
261 171 77
319 183 157
THE APPLICATION NOTEBOOK -- September 2004
Results and Discussion A typical matrix-matched calibration curve was linear in the range from 0.1 to 10 g/L using 20-mL samples of river water. Limit of quantitation is below 0.2 g/L. Figure 2 shows a chromatogram obtained from a 0.5-g/L spiked sample using HILIC chromatography. A series of 1-g/L (1 ppb) spiked samples was analyzed over a five-day period, five replicates/day. The results are summarized in Table II.
Although LCMS might be preferable for this analysis, the Oasis® WCX SPE protocol is certainly compatible with ion-pair LCUV analysis. Figure 3 shows a typical separation for a 0.5-g/L spiked sample using ion-pairing chromatography on an Atlantis® dC18 column. However, the ion-pairing method should not be used for high sensitivity LCMS analysis. The inset (Figure 2) shows the relative LCMS response obtained for the same standard using the HILIC method compared with the ion-pairing method.
Table II: Intraday results obtained from spiked river water samples
(spike level 1.0 g/L)
Day 1 1.08 g/L (8.1% RSD) Day 4 1.10 g/L (8.0% RSD) Day 5 0.95 g/L (7.1% RSD) Overall (n 15) 1.04 mg/L (9.8% RSD)
Day 1 1.05 g/L (2.9% RSD) Day 4 1.09 g/L (5.9% RSD) Day 5 1.08 g/L (4.4% RSD) Overall (n 15) 1.08 mg/L (4.8% RSD)
Figure 2: LCMS chromatogram showing the separation of paraquat and diquat at 0.5 g/L using the Atlantis HILIC analytical column. Inset shows the relative LCMS response for HILIC compared with ion-pair reversed-phase chromatography.
Figure 3: LCUV chromatogram showing the separation of paraquat and diquat at 0.5 g/L using the Atlantis dC18 analytical column. The aqueous mobile phase contained 0.1% heptafluorobutyric acid as ionpairing reagent.
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MS Young, KM Jenkins