Methods of residue analysis

Analytical methods

The 1993 JMPR monograph describes methods which have been used for the residue analysis of samples from crops and animals. The methods summarized in the 1993 monograph and those submitted to the present Meeting are tabulated below. References are given in the text.

Method	Year	Limit of determination, mg/kg	Detector	Substrates
1993 JMPR
REM 7a/73	1973	0.02	FID	Apple, lettuce, bean leaves
REM 15/82	1982	0.02	NP	Cherry, lettuce, cocoa seed
REM 119.01	1989	0.01	EC or NP	Kiwifruit, maize (whole plant)
AG-550A	1990	0.01-0.021 diazinon, diazoxon	FPD	(21 crops, almonds, corn oil, animal tissues
		0.021 hydroxydiazinon
		0.05 ditto		Hops
	Generally 0.02 mg/kg may be a more practical limit of determination for diazinon and metabolites in animal products via AG-550A, although 0.01 mg/kg may be attainable in some cases.
1996 JMPR
Method 113	Undated	0.02	Thermionic	Sheep tissues and fat
Method 29/73	1973	0.02-0.051	AFID	Meat and milk
Method 4/74	1974	0.021 diazinon	FPD	Animal tissues
		0.051 hydroxydiazinon
		0.21 G-27550
REM 21/86	1987	0.021	NPD	Muscle, liver, kidney, fat
Netherlands Official Methods	1988	0.01-0.05	NPD	Crops
		0.01-0.04		Meat, tissues
		0.001-0.01		Milk
REM 128.02	1991	0.02'	NPD	Milk
Method 132A	1994	0.01	NPD-thermionic	Milk
Method 132B	1992	0.01-0.02'	N-P	Butter
Method 135	1994	0.01	thermionic	Muscle, liver, fat

¹ Estimate by the present Meeting (often twice the reported value). In all other cases the reported value could not be confirmed with information provided. LODs apply to diazinon unless otherwise indicated.

Method AG-550A, which has been used extensively for animal products, is discussed below. Other methods summarized by the 1993 JMPR will not be described again.

Method AG-550A (Hubbard et al., 1990) involves extraction of crops and various animal tissues and milk with acetone/water, partitioning into petroleum ether/methylene chloride, concentration, dissolution in acetone and GLC with a flame-photometric detector. It was tested on 21 crops. Some variation is provided for selected samples. For hops the solvent is evaporated and the residue dissolved in hexane and partitioned into acetonitrile before evaporation of the acetonitrile and transfer to acetone for analysis. Corn oil is extracted directly with acetonitrile which is similarly evaporated and the residue transferred to acetone. Beef fat is extracted with hexane before partitioning into acetonitrile, otherwise its treatment is similar to corn oil. An alternative mini-Florisil column clean-up is provided to remove material which interferes with GLC.

Method AG-550A was used in most of the US trials. More importantly, it was the method used in the animal transfer studies reviewed here, in which it was used to determine diazoxon and hydroxydiazinon as well as diazinon. Generally a limit of determination of 0.01 mg/kg (0.05 mg/kg in hops) is reported to be achievable for diazinon and the metabolites with use the preferred capillary columns, and 0.025 mg/kg for diazinon and 0.05 mg/kg for the metabolites with packed columns.

The validations and sample chromatograms provided were generally consistent with the estimated limits of determination, with analytical recoveries generally >96% for all three compounds in meat, eggs, fat and milk at a fortification level of 0.01 mg/kg. The exception was 68% for the determination of diazoxon in beef liver. Recoveries were similar in the transfer studies, but again a little lower in liver and kidney. The method was also validated (Hubbard, 1990) for crops and animal tissues with [¹⁴C]diazinon in fortified samples and in goat tissues from animal metabolism studies.

Representative chromatograms in the animal transfer study by Selman (1994a) also suggest that 0.01 mg/kg of all three compounds can probably be determined in milk and tissues, although the results were not as convincing with kidney and fat, especially for hydroxydiazinon for which 0.02 mg/kg would appear to be a more reasonable limit of determination. Also on the basis of sample chromatograms in poultry transfer studies (Selman, 1993) 0.02 mg/kg may be a more practical limit of determination, especially for hydroxydiazinon. Measurement with confidence at 0.01 mg/kg may be possible, however, at least for fat and eggs.

Method 113 (Anon., undated) determines diazinon in the fat and tissues of sheep and is based on extraction with hexane, sweep co-distillation and determination by GLC and a thermionic detector. A "method sensitivity" of 0.02 mg/kg was reported, but no information on validation or sample chromatograms were provided.

REM 29/73 (Formica, 1973) for diazinon involves extraction of meat with methanol and milk with acetone, partitioning into chloroform, clean-up on an alumina column and determination by GLC with either flame-photometric or alkali flame-ionisation detectors. The reported limit of detection was 0.01 mg/kg in meat and milk. Recoveries were >94% at fortification levels of 0.03 mg/kg in milk and 0.05 mg/kg in meat. Because sample chromatograms of controls showed no really quantifiable residues and since the method was not validated below 0.03 or 0.05 mg/kg, a limit of determination of 0.02-0.05 mg/kg should be achievable.

REM 4/74 (Formica, 1974) was developed for the determination of diazinon, diazoxon, hydroxydiazinon and 4-hydroxy-2-isopropyl-6-methylpyrimidine (G 27550) in animal tissues. For the determination of diazinon, hydroxydiazinon and G 27550, the tissues are macerated or extracted with methanol, the extract is diluted with 1 N HCl and diazinon and hydroxydiazinon are extracted with chloroform. G 27550 is likewise extracted with chloroform after neutralization of the HCl. Diazinon and hydroxydiazinon are cleaned up on an alumina column (or by TLC) before GLC analysis with an FPD. G 27550 is determined with a nitrogen-selective electrolytic conductivity detector. Diazoxon is determined by cholinesterase inhibition, but as this is not a currently acceptable method it will not be further described.

The lowest levels at which analytical recoveries were measured were 0.1 mg/kg of diazinon and hydroxydiazinon and 0.2 mg/kg of G 27550, at which levels recoveries were generally >75% from sheep muscle, liver and fat and sow kidney and liver, but only 62% of G 27550 from sheep fat. The limits of detection of diazinon and hydroxydiazinon were reported as 0.01 and 0.02 mg/kg respectively. For G 27550 an interfering GLC peak resulted in a reported limit of detection of 0.1 mg/kg. From sample chromatograms and the validation levels, reasonable limits of determination in muscle would appear to be about 0.02 mg/kg for diazinon, 0.05 mg/kg for hydroxydiazinon and 0.2 mg/kg for G 27550. In the absence of sample chromatograms, the Meeting could make no estimates for other tissues.

In REM 21/86 (Netherlands, 1988) diazinon is extracted from homogenized muscle, liver or kidney with methanol, partitioned into hexane, and cleaned up on a phenyl-coated solid-phase extraction column. Heated fat is extracted with acetonitrile, the extract partitioned with hexane for clean-up, and the acetonitrile rotary-evaporated. The extract is taken up into hexane and cleaned up on a cyano-coated solid-phase extraction column. Determination is by GLC with an NP detector.

Analytical recoveries were >85% at a fortification level of 0.02 mg/kg from liver, kidney and fat. The limit of determination was reported to be 0.01 mg/kg. Sample chromatograms suggest that 0.02 mg/kg may be a more practical limit for sheep muscle and fat. Sample chromatograms were not provided for liver or kidney, so the Meeting could not estimate LODs for them.

The Netherlands Official multi-residue GLC Sub-method 1 (1988) for diazinon in fruits and vegetables involves extraction with ethyl acetate and analysis by GLC with a phosphorus-specific detector without further clean-up. For crops recoveries of 80% (fortification level unspecified) and limits of determination of 0.01-0.05 mg/kg are reported. In Sub-method 2 for the determination of diazinon in animal tissues extraction with acetone/acetonitrile is followed by evaporation, partitioning into acetonitrile from hexane and determination by GLC with an NPD. Recoveries of 66-102% and limits of determination of 0.01-0.04 mg/kg are reported. In Sub-method 3 for diazinon in milk, extraction with ethyl acetate is followed by evaporation, dissolution in hexane, partitioning with acetonitrile, evaporation, solution in ethyl acetate and analysis by GLC with a phosphorus-specific detector. Recoveries of 75-100% and limits of determination of 0.001 to 0.01 mg/l are reported. Recoveries and limits of determination were not included in the submission to the JMPR.

REM 128.02 (1991) was developed for the determination of diazinon in blood and milk. The method for milk is based on Method AG-550A, but the initial acetone/water extract is cleaned up on a C-18 solid-phase cartridge before GLC determination with an NPD. The "lower practical level" for milk by this method was reported as 0.008 mg/kg. At the lowest fortification level of 0.02 mg/kg the average recovery from milk was 104%. A sample chromatogram in the report suggested a general practical limit of determination of 0.02 mg/kg, although 0.01 mg/kg might be achievable.

Method 132A (1994) for the determination of diazinon in milk is similar to AG-550A, but residues are extracted with acetone and transferred to methylene chloride, which is evaporated. The residue is taken up into hexane, partitioned into acetonitrile, concentrated into added methanol and finally cleaned up on an alumina column. A recovery of 86% is reported at the lowest fortification level in the report (0.1 mg/kg). The limit of determination is reported to be 0.01 mg/kg, although sample chromatograms of controls and fortified samples were not available for independent confirmation.

Method 132B (1992) determines diazinon in butter. The butter is dissolved in hot hexane and the residues partitioned into acetonitrile, which is evaporated to dryness. Clean-up is on an alumina column and determination by GLC with an NP detector. The average recovery was 90% at the lowest fortification level (0.02 mg/kg). A limit of determination of 0.01 mg/kg is reported and a sample chromatogram indicates that that should be achievable.

Method 135 (1994) determines diazinon in fat and animal tissues. Muscle and liver are macerated with methanol, which is diluted with water and extracted with methylene chloride. This is evaporated and the residue cleaned up on an alumina column. Fat is ground with sodium sulfate, extracted with hot hexane, and cleaned up by partitioning into acetonitrile. The acetonitrile is evaporated and the residue taken up in hexane and further cleaned up on an alumina column. Determination is by GLC with thermionic detection. Analytical recoveries of 84% from fat, 90% from muscle and 82% from liver were reported at the lowest fortification level of 0.1 mg/kg. The limits of detection and determination were reported to be 0.01 mg/kg, but the report did not include sample chromatograms of controls and fortified samples for independent confirmation.

Stability of pesticide residues in stored analytical samples

The studies of storage stability in crops and processed commodities (Beidler and Moore, 1991) and animal tissues (Schnabel and Formica, 1981) reviewed and described by the 1993 JMPR were re-submitted. From the latter study the 1993 JMPR noted that diazinon residues in animal tissues were stable for at least 8 months. The study was on samples from sheep which had been dipped in diazinon and contained initial residues of 0.05-2 mg/kg in muscle, <0.1 mg/kg in liver, 0.08-0.5 mg/kg in kidney and 2.8-5 mg/kg in fat. Samples were stored at -20°C. The study did not include information on the storage stability of metabolites in tissues, or of diazinon or metabolites in milk.