Analytical Techniques

1. Qualitative confirmation of diagnosis

1.1 TEST TUBE TEST

• Urine or gastric aspirate can be tested for paraquat using the method based on reduction of paraquat cation to a blue radical ion in the presence of alkali and sodium dithionite
  
• Add alkali, such as sodium hydroxide, to 10 ml or urine or gastric aspirate until the pH is above 9 (approximately half to one teaspoon of sodium bicarbonate can be used as an alternative).
  
• Add a spatula blade full of sodium dithionite to the alkaline urine or gastric aspirate and mix gently.
  
  NB. Once opened and exposed to air/moisture sodium dithionite can deteriorate on storage so users should ensure that the reagent works effectively preferably by testing a sample known to contain paraquat. Sodium dithionite packed in foil sachets supplied in test kits has a shelf-life of at least 10 years if undamaged.
  
• View the tube from above against a white background. A blue or green colour in the solution denotes the presence of paraquat and confirms the diagnosis. In the presence of high paraquat concentrations, the solution may turn black, and the test should be repeated with a diluted sample.
  
• This method can detect concentrations of paraquat in urine down to 2 µg/ml and may be made semi-quantitative if a range of standards are prepared in control urine samples. (Widdop 1976; Berry and Grove, 1971).

1.2 SOLID PHASE EXTRACTION

Urine, serum or plasma can be tested for paraquat using a method based on in situ reduction on a solid phase extraction cartridge ( Woollen and Mahler 1987). A more sensitive version of this test using the reagents provided in the test kits is detailed below.

Plasma can block the cartridges so if possible it should be filtered, for example through a 0.45µm syringe filter ( PVDF or nitrocellulose ) before performing the test. Serum does not require this pre-treatment unless it is cloudy.

• Mix approximately 1g portions of sodium bicarbonate and sodium dithionite with 10 mL water and allow to settle.
• Urine is made alkaline by addition of approximately 0.5g of sodium bicarbonate to 5 mL urine.
• Transfer 2 mL plasma, serum or alkalinised urine to a 1 mL/100mg silica SPE cartridge, and allow the sample to percolate through into the bed (the preferred cartridge is Bakerbond Cat No 7086-01, an alternative is Varian Bond-Elut 14102010).
• Using a syringe fitted on to the top of the cartridge with an adapter, apply gentle pressure to force the rest of the sample through the cartridge.
• Wash the cartridge with an equal volume of water, keeping the flow rate low.
• Add approximately 0.2 mL of the dithionite solution to the cartridge and apply very slight pressure to ensure that the liquid penetrates to just below the top frit. Do not allow the cartridge to dry out
• A blue band immediately below the top frit of the cartridge indicates the presence of paraquat and confirms the diagnosis.
• This test will detect paraquat down to a level of around 0.1 µg/ml with a 2 mL sample. Ideally a positive control should be carried out, at a level of around 0.5 µg/mL.

2. Quantitative determination of paraquat

Quantitative measurement in plasma gives a measure of severity and prognosis (the sample must be taken at least four hours post-ingestion, and should be centrifuged and stored in plastic, not glass tubes.)

2.1. SPECTROPHOTOMETRY FOLLOWING SOLID-PHASE EXTRACTION AND REDUCTION WITH SODIUM DITHIONITE

• Plasma or serum samples are first filtered as described in section 1.2. Bond-Elut cyanopropyl cartridges (100mg 1 mL reservoir, Varian) are pre-conditioned successively with two column volumes of methanol, 0.1M HCl and 0.1M ammonia solution. The cartridges are fitted with 15 mL reservoirs and the patient plasma, serum, blank or spiked plasma ( 5 mL) is applied and drawn through by attaching the cartridges to a vacuum manifold until the cartridges are dry. The cartridges are rinsed with 1 mL 0.1M ammonia until they are dry and then paraquat is eluted with 0.8 mL 0.1M HCl into a test tube. Concentrated ammonia (0.025 mL) and sodium dithionite (0.1 mL 0.23M in 4M NaOH) are added. After vortexing the solutions are poured into disposable semi-microcuvettes (1 mL) and the absorbance scanned from 490 to 385 nm. The absorbance difference (A395-A460) is used to determine the paraquat concentration.
• A standard curve is prepared in the range 0.05 - 1µg/mL paraquat ion. Higher concentrations in unknown samples may be determined by taking a smaller amount of plasma sample. The lower limit of quantitation of the assay is 0.045 µg/ml based on a 5 mL sample.
• The method is also applicable to urine which must be alkalinised (0.025 mL concentrated ammonia to 5 mL urine) and centrifuged prior to addition to the cartridge.

2.2. HPLC FLUORESCENCE

• Paraquat can be measured in plasma or urine down to 0.001µg/mL by fluorescence HPLC after conversion to the dipyridone derivative (Blake, et al 2002).

3. Advice on paraquat analysis

Syngenta CTL can provide advice on analysis of paraquat in biological samples via the e-mail address ctltestkitsupply@syngenta.com