Cryogenic Sample Processing with Liquid Nitrogen for

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Perspective

Cryogenic Sample Processing with Liquid Nitrogen for Effective and Efficient Monitoring of Pesticide Residues in Foods and Feeds Manol Roussev, Steven J. Lehotay, and Julius Pollaehne J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b04006 • Publication Date (Web): 01 Aug 2019 Downloaded from pubs.acs.org on August 1, 2019

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Journal of Agricultural and Food Chemistry

Cryogenic Sample Processing with Liquid Nitrogen for Effective and Efficient Monitoring of Pesticide Residues in Foods and Feeds Manol Roussev,a,* Steven J. Lehotay,b and Julius Pollaehne a a WESSLING

GmbH, Haynauer Strasse 60, D-12249 Berlin; Germany

b

United States Department of Agriculture, Agricultural Research Service, Eastern Regional Research Center; 600 East Mermaid Lane; Wyndmoor, Pennsylvania 19038; USA * To whom correspondence should be addressed. Tel: +49 30 77 507 401; Fax: +49 30 77 507 555; E-mail: [email protected] Disclaimer: The use of trade, firm, or corporation names does not constitute an official endorsement or approval by the USDA of any product or service to the exclusion of others that may be suitable. Note: The authors declare no competing financial interest. Graphical Abstract:

ABSTRACT: 1

When monitoring hundreds of pesticides in food and feed, the comminution step is equally

2

crucial as any other to achieve valid results. However, sample processing is often

3

underestimated in its importance and practical difficulty to produce consistent test portions for

4

analysis. The scientific literature is rife with descriptions of micro-extraction methods, but

5

ironically, sample comminution is often ignored or dismissed as being prosaic, despite that it

6

is the foundation upon which the viability of such techniques relies. Cryogenic sample

7

processing using dry ice (-78°C) is generally accepted in practice, but studies have not shown 1

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8

it to yield representative test portions < 1 g. Remarkably, liquid nitrogen has rarely been used

9

as a cryogenic agent in pesticide residue analysis, presumably due to access, cost, and safety

10

concerns. Yet, real-world implementation of blending unfrozen bulk food portions with

11

liquid nitrogen (-196°C) using common food processing devices has demonstrated this

12

approach to be safe, simple, fast, cost-effective, and yield high-quality results for various

13

commodities, including increased stability of labile or volatile analytes. For example,

14

analysis of dithiocarbamates as carbon disulfide has shown a significant increase of thiram

15

recoveries (up to 95%) by using liquid nitrogen during sample comminution. This

16

perspectives article is intended to allay concerns among working laboratories about the

17

practical use of liquid nitrogen for improved sample processing in the routine monitoring of

18

pesticide residues in foods and feeds, which also gives promise for feasible test sample size

19

reduction in high-throughput miniaturized methods.

20 21 22 23

KEYWORDS: comminution, cryogenic sample processing, liquid nitrogen, pesticide residue analysis, foods

24

INTRODUCTION

25

In 2015, Lehotay and Cook authored a Perspectives article in this Journal to highlight

26

the essential importance of sampling and sample processing (comminution) in pesticide

27

residue analysis.1 A main point bears repeating: “If collected samples and test portions do

28

not adequately represent the actual lot from which they came and provide meaningful results,

29

then all costs, time, and efforts involved in implementing programs using sophisticated

30

analytical instruments and techniques are wasted and can actually yield misleading results.”

31

In the past four years, further investigations of this topic have been reported,2-8 and additional

32

practical information about cryogenic sample processing using liquid nitrogen has come to

33

light that warrants this updated Perspectives article to describe new developments, practical

2


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implementation, and possibilities for its use in novel miniaturized high-throughput

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applications.

36 37

CRITICAL REVIEW

38

As detailed previously,9 analytical chemists tend to focus on techniques aimed to improve

39

chemical separation and detection methods but ignore or minimize the equally important

40

sample processing step. Although 100% of chemical analyses entail sample processing in

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some manner, only 0.3% of publications on food analysis even mention common terms on the

42

subject in the title, abstract, or keywords - compared with 13% of papers in the case of

43

spectrometry or spectroscopy, for example.7

44

A literature search using Web of Science was conducted in April of 2019 to peruse the

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200 most recent publications about pesticide residue analysis at the time. As before,9 a

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notable number of papers involved (bio)sensors,10-11 nanotechnology,12-15 surface-enhanced

47

Raman spectrometry (SERS),14-16 and other micro-extraction techniques. A disproportionate

48

number of publications entailed analysis of liquid samples (e.g., water, juices, wine) rather

49

than food or soil samples, undoubtedly in part due to greater ease of sample processing and

50

preparation of liquids vs. solids. However, even liquids such as juices are not so

51

homogeneous that micro-samples are likely to be representative of larger volumes.6 Watery

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matrices also tend to be much cleaner to yield better analytical performance and robustness

53

than complex foods that contain high percentages of proteins, carbohydrates, and/or lipids.

54

Clearly, some investigators adjust the application to meet the limitations of a novel analytical

55

tool rather than devise the tool (or choose an approach) to meet the needs of the application

56

for a real purpose. Sometimes, publications are the sole purpose for analyses, and if an

57

underlying requirement for valid sample processing is unmet, that issue is usually ignored or

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dismissed.

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Several examples could be listed, but only a few recent papers will be cited to make

60

the point. Wei et al.17 analyzed 1 g test portions by solid-phase microextraction (SPME) of an

61

unknown amount of oranges “homogenized by a high-speed food blender.” Although

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comminuted oranges have been found to be more homogeneous than other commodities,18-19

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cryogenic sample processing is still needed to achieve accurately representative 1 g

64

subsamples of the original bulk sample.4-5,7-8,18-19 In other examples of micro-extraction,

65

Gorji et al.20 analyzed 0.3 g test portions of rice, cucumber, and tomato, and Xue et al.21 used

66

0.2 g samples for chrysanthemum.

67

Independent of any practical advantages or improved quality of results described in

68

such studies, the methods are impractical in real-world implementation unless the sample

69

processing procedures for the analytes and matrices are also demonstrated to be valid for the

70

analyzed test sample portions. For regulatory and other purposes, typically > 500 g bulk

71

samples need to be comminuted, and subsamples need to represent the original sample.1,22

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However, studies have already shown that < 10 g subsamples using typical sample processors

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at ambient conditions frequently leads to unacceptably high variability and bias for tested

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pesticides and foods.1-8,18-19 Cryogenic comminution is needed for improved results for

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smaller test portions, and Riter et al.5 demonstrated that some pesticide/sample pairs still yield

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excessive error for < 1 g subsamples even when using specialized instrumentation and

77

techniques. In practice, Han et al.7 failed in their attempts to conveniently and consistently

78

weigh 0.25-0.5 g food portions in their experiments, independent of how well the original

79

sample was homogenized.

80

Therefore, known science should already dissuade researchers from analyzing

81

unrepresentatively small subsamples in routine pesticide residue applications. Logic and

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common sense dictate that researchers should also focus their efforts to improve the sample

83

processing step when they are developing yet another method among hundreds already

84

published that involve microsamples. Those genuinely interested in the dissemination of 4


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micro-techniques, commercialization of new products, and improving the state of pesticide

86

residue analysis in general would recognize that the limitations in sample processing must be

87

overcome to enhance the viability of their approaches. The successful development and

88

demonstration of a practical approach that solves this problem for many analytes and matrices

89

would apply to hundreds of currently infeasible miniaturized methods, which should lead to

90

highly-cited publications about such a broadly implementable breakthrough.

91

Despite that sample comminution is even more critical in micro-extraction techniques

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than traditional methods, this issue is essentially being ignored. In a review entitled

93

“Applications of [SPME] with mass spectrometry in pesticide analysis,” sample processing is

94

not even mentioned.23 Too few investigators and reviewers bother to consider the prosaic

95

topic of sample processing, or hold themselves and others accountable to develop practical

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methods that meet real-world needs, but the weak link of sample comminution in the

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analytical chain is not so easy to strengthen,9 which likely explains why it is largely ignored in

98

the first place.

99 100

REGULATORY CONSIDERATIONS

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Fussell wrote an excellent primer about the global issue involving regulation of pesticide

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residues in food.24 Sampling and sample processing are essential components to comply with

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analytical quality standards to assure that maximum residue levels (MRLs), or tolerances in

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the US, for pesticides in food and feed are not exceeded. An accurate determination of active

105

substances and/or their metabolites is needed for enforcing laws, regulating food trade,

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assuring that producers are not harming the environment or consumers with improper usage of

107

pesticides, or misrepresenting their food as being grown organically if it is not. Millions of

108

dollars are often at stake in pesticide analyses in foods, and incorrect results can be very

109

damaging to all parties involved.

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In the USA, an extensive technical overview and guidance document was recently

110 111

published to help labs conduct proper sample processing for analysis of foods and feeds.6 In

112

the EU, the “Guidance document on analytical quality control and method validation

113

procedures for pesticide residues and analysis in food and feed” (SANTE/11813/2017) also

114

makes recommendations about sample processing, including the suggestion that cryogenic

115

conditions generally improve the quality of the analysis.25 Codex Alimentarius is another

116

body that has published international recommendations about sample processing for pesticide

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residue analysis.22 The EU report, “Technical guideline on the evaluation of extraction efficiency of

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residue analytical methods,” highlights the concern with poor extraction efficiencies of

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incurred pesticides in real samples vs. those spiked into blank matrices in validation

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procedures.26 Because the actual concentrations of incurred residues are unknown, a better

122

assessment of analytical methods is needed through the use of radioactive isotopes, repetitive

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extractions, analysis of reference materials, and/or comparison of results from shared samples

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using different methods. In the latter case, proficiency testing is required for accreditation by

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ISO 17025:2017 standards,27 but the shared test samples are already comminuted for the

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participants; thus the sample processing step is not included in the comparison of results. Since the quality of laboratory results also depends on sample processing, this step in

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the method should also be evaluated.1,2,6-9 Studies have shown that proper sample

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comminution contributes less than half of the overall measurement uncertainty in common

130

analyses, but the use of excessively small subsamples or improper procedures can lead to

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systematic and random errors that constitute 100% of the overall uncertainty. 1-8,18-19 The

132

assumption cannot be made that “proper” sample processing is being done in analyses. Thus,

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quality control measures should be taken to verify the performance of this initial step in the

134

method, 1-9 just as commonly done to cover the subsequent sample preparation and analysis

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steps. 6


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CRYOGENIC COMMINUTION

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Cryogenic sample comminution has been demonstrated to achieve much better homogeneity

139

for smaller subsample sizes and reduced degradation of labile analytes,4-5,18-19 but it also adds

140

to the time, cost, and labor to conduct sample processing compared to ambient comminution.

141

Simply chopping the bulk samples at room temperature with a food processor can achieve

142

relatively high sample throughput and acceptable results for typical pesticide commodity

143

combinations using ≥ 5 g subsamples7-8 (for analytes that are not labile or volatile). Although

144

Riter et al. describe high-throughput sample processing using a two-step cryomilling method

145

using liquid nitrogen to yield consistently representative 75 mg test portions for glyphosate

146

analysis,4-5 their throughput of 50 samples per person per day using multiple devices may still

147

not meet desired lab throughput needs, and the technique requires relatively high cost and

148

skill.

149

What other options are available? Cryogenic milling using dry ice (-78°C) is

150

generally accepted for improving sample homogeneity and pesticide stability during the

151

comminution procedure.1,18,19,25 However, a significant disadvantage of using dry ice is that

152

the bulk samples need to be diced and frozen prior to the addition of the dry ice or else an

153

eruption of foggy CO2 gas (and bits of sample) will result from the food processor. Also, dry

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ice takes many minutes to sublime after it has mixed with the sample. Thus, additional time is

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needed for the sample weight to stabilize before an accurately representative test portion can

156

be taken. Some routine monitoring labs spend two days to process samples using dry ice due

157

to the extra time needed for cutting, pre-freezing, and post-sublimation of samples.

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Moreover, condensation of water from the air occurs during cryogenic processing in humid

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environments, which can lead to a bias in the results, particularly when < 2 g subsamples are

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used as test portions.7,8

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Most critically, condensation of water from the atmosphere occurs on the dry ice itself

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before it is added to the sample. This water is then added to the sample during comminution,

163

leading to a bias in the ensuing results. The purchased dry ice may be stated to be 99.99%

164

pure, for example, but water condensation may reduce that purity during storage, and the

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purity is further reduced each time the dry ice is exposed to a humid atmosphere. Lastly, a

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clean source of dry ice must be available, and safe practices must be followed to avoid

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possible frozen skin burns and suffocation in an unventilated, enclosed space.

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Liquid nitrogen (-196°C) is a superior cooling agent vs. dry ice, but it is rarely used for

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comminution in pesticide residue analysis. Safety concerns are more severe for liquid

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nitrogen than dry ice, but similar types of safety precautions must be taken in many routine

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lab procedures. Working in ventilated hoods with cryogenic gloves and a face shield is

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standard practice when chemists pour liquid nitrogen from Dewars, but the use of valving

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systems in well-ventilated labs avoid these inconveniences. Placing oxygen sensors with

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alarms in areas where liquid nitrogen is employed also serves as an appropriate precaution.

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In terms of access, many facilities use liquid nitrogen as the primary source for

176

nitrogen gas, which is commonly required for mass spectrometers, for example, and NMR

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instruments usually need liquid nitrogen directly. At one of the author’s facilities, the cost of

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bulk purchases of >99.998% pure liquid nitrogen is about $0.15 (US) per kg vs. $0.75 per kg

179

for pelletized dry ice. Liquid nitrogen generators can be purchased to further lower long-term

180

expenses. Moreover, the liquid nitrogen remains pure in closed tubing or Dewars, and even in

181

open vessels, condensed water freezes and sinks to the bottom rather than mix with the liquid

182

nitrogen, unlike the case with dry ice. Thus, water is not introduced into the sample when

183

liquid nitrogen is poured into it.

184

Another advantage of liquid nitrogen over dry ice is that a small amount of carbonic

185

acid is formed when CO2 is mixed with water, which can change the pH of the sample and

186

induce undesired effects in the analytical methods. By contrast, N2 is largely unreactive. 8


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USE OF LIQUID NITROGEN IN PRACTICE

189

About 10 years ago, the WESSLING lab in Berlin (Germany) successfully evaluated liquid

190

nitrogen to replace dry ice in sample comminution of bulk commodities > 500 g for analysis

191

of 10 g test portions in the QuEChERS method. Applying liquid nitrogen instead of dry ice

192

usually allows immediate comminution of unfrozen, uncut bulk commodities. Commodities

193

with large individual units, such as apples, melons, etc., need to be cut into smaller sections in

194

any case,6,22,25 but they can be cryogenically processed immediately using liquid nitrogen,

195

unlike with dry ice. Also, the nitrogen converts to room temperature gas quickly in a way that

196

does not lead to an eruption of foggy vapor mixed with bits of the sample, if the appropriate

197

amount of liquid nitrogen is employed.

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In practice, stainless steel vessels should be employed without any plastic parts that

199

come into contact with the liquid nitrogen. Also, briefly precooling the vessel (including

200

blades) in a freezer prior to comminution helps reduce nitrogen usage that would otherwise be

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needed to cool the container. Similarly, precooling of metal spatulas or spoons avoids

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thawing and stickiness of the frozen powdery samples when taking test portions. Cutting

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blades will need to be sharpened more often when using liquid nitrogen, but this does cause a

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practical inconvenience. For raw commodities at room temperature, such as strawberries, the

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WESSLING lab has found that ≈1.5 L liquid nitrogen per kg sample is sufficient for good

206

operation, and much less is needed in the case of dry samples (cereals, tea, etc.). The use of

207

excessive liquid nitrogen does not pose a problem with comminution, but use of too little can

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lead to undesirable thawing and agglomeration of sample particles.

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In a single step, 10 g test portions are taken for QuEChERS sample preparation,28

210

including determination of dithiocarbamates as CS2.29 This approach achieves high sample

211

throughput of 10 samples per person per hour. Although extraction of a smaller subsample

212

size has not been a goal of the lab, we hypothesize that direct comminution of the sample 9



213

immersed in liquid nitrogen using contemporary food processors, as shown in Figure 1, can

214

yield accurately representative test portions < 1 g if desired. We encourage others to

215

investigate this issue for possible application in their micro-sample extraction methods.

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Unlike indirect use of liquid nitrogen as in cryomill devices,4-5, 7-8 direct immersion of

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the typically uncut commodities with liquid nitrogen in a sufficiently large bowl freezes the

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sample more quickly and efficiently in a single step, using much lower cost of equipment and

219

less liquid nitrogen. Furthermore, currently commercially available cryogenic devices using

220

liquid nitrogen need < 25 g sample sizes in practice with a maximum of two parallel samples

221

per device, which also involves skilled manual sample handling. The programmed

222

comminution methods using the cryomill devices also takes several minutes, whereas the

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direct use of liquid nitrogen in standard inexpensive food processors takes < 3 min per

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sample. Riter et al. required a two-step homogenization procedure using 3 rather expensive

225

cryomill devices to process ≈6 bulk samples/person/hour.4-5

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Not only does comminution with liquid nitrogen increase sample homogeneity, but it

227

also produces smaller particle sizes for better access of the extraction solvent to the samples

228

(therefore improved extraction efficiency).1,6 This is more important for analysis of incurred

229

samples than spiked samples, as discussed previously.26 Notably, if sample processing of

230

proficiency test samples or reference materials does not produce sample particulates as fine as

231

needed for complete extraction efficiency, then bias in the results will occur. Comminution

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with liquid nitrogen reduces this possibility.

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Water condensation from a humid laboratory environment into the sample is a concern

234

with any cryogenic method. The extent of condensation on sample weight was tested in an

235

experiment using liquid nitrogen comminution, and Table 1 lists the results. A laboratory mill

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GM 300 (Retsch; Haan, Germany) with 4.5 L vessel volume was used per usual at the

237

WESSLING lab. In an experiment, a series of 5 bulk strawberries samples of ≈ 650 g each

238

were comminuted with liquid nitrogen for 90 s, and then for another 90 s, and reweighed each 10


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time (in routine operations, the WESSLING lab processes > 1,000 g bulk samples in

240

accordance with Directive 2002/63/EC). The maximum weight increase was < 2% of the

241

original sample in the first 90 s, but this was found to be solely due to condensation on the

242

outside of the container, as shown in the picture associated with Table 1. When the container

243

was wiped to remove the water after a total of 180 s, no appreciable difference in sample

244

weight was measured. In fact, the closed (but not sealed) container barely allows atmospheric

245

air or moisture to access the sample because the nitrogen flows outward, leaving the container

246

in nitrogen-rich, dry atmosphere. The water in the sample remains frozen, thus does not

247

readily evaporate. Similarly, the liquid nitrogen dissipates immediately from the sample,

248

unlike dry ice, which can take many minutes to sublime.

249

Working quickly to weigh the test sample portions into extraction tubes also helps to

250

reduce biases due to addition or loss of moisture. Test portions can be analyzed immediately

251

or stored in sealed vessels in a freezer. In the case of dry ice, the released CO2 gas from the

252

remaining dry ice in the sample may act to pressurize a sealed vessel, thus time is usually

253

given before the caps are sealed tightly in that case.

254

The rapid freezing process with liquid nitrogen causes the sample to shatter easily due

255

to its brittleness, and the comminuted samples quickly turn to a fine powder in the container,

256

as shown in the images to the right in Figure 2. The traditional sample processing approach

257

leads to visibly worse samples with which to work, as shown on the left in Figure 2. At the

258

top, cocoa beans and similarly hard low moisture commodities often pose difficulties for

259

common food choppers or laboratory mills at room temperature, but freezing even 1-2 kg of

260

such samples with liquid nitrogen renders the commodities rapidly and easily comminuted

261

into a fine powder using the same type of chopper. Liquid nitrogen also significantly eases

262

the processing of complex matrices, such as tea, herbs, and roots, which make comminution a

263

simple routine with little difference among commodities, unlike in traditional procedures.

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As indicated at the bottom of Figure 2, high-sugar and low-water containing

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commodities (e.g., dried fruit) are commonly comminuted in choppers by adding water to

266

obtain slurries. However, applying liquid nitrogen obviates the need to add water before

267

comminution, and samples such as raisins or cranberries become free-flowing and powdery

268

(Figure 2). Note: the condensation shown in the cranberry powder resulted from the time

269

taken to prepare the sample for the picture, and is avoided in routine practice. Another

270

advantage of comminuting the dry sample rather than adding water to the bulk sample is that

271

weighing the dry test portion into the extraction tube is more accurate than weighing a less

272

homogeneous slurry that lacks a uniform amount of original sample vs. added water. In the

273

former case, an exact volume of water is consistently added to an exact amount of dry test

274

portion each time, which improves extraction.

275 276

ANALYTE STABILITY

277

Liquid nitrogen provides an exceedingly cold and inert environment to reduce evaporative

278

and degradative losses of highly volatile and/or labile pesticides in commodities.

279

Furthermore, enzymatic and other reactions are essentially terminated at liquid nitrogen

280

temperature when the pesticides are being mixed with and exposed to reactive components

281

within the sample matrix or on container surfaces. Indeed, in some cases, addition of acid

282

using ambient comminution is helpful for analyte stability. For example, tolylfluanid and

283

dichlofluanid in cucumber using ambient comminution were recovered at only 2% and 7%

284

without addition of acid, whereas milling in acidic conditions yielded > 75% recoveries for

285

both pesticides (regardless of using dry ice). However, in the case of thiodicarb, adding acid

286

lead to poor recovery of 5% and only milling under cryogenic conditions significantly

287

increased the recovery to 83%.30

288

As described in the EU SANTE guidance document on analytical quality control,25

289

“where comminution is known to affect residues (e.g. dithiocarbamates…) the test portion 12


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should consist of whole units of the commodity, or segments removed from large units.”

291

More specifically,29 “Dithiocarbamate residues are typically located superficially. Thus,

292

sample comminution (e.g. cutting, milling, grinding) is only to be performed, where this is

293

necessary to obtain acceptable sub-sampling variability.” The protocol further calls for

294

storage of the sample overnight in a freezer followed by cryogenic comminution with dry

295

ice.29

296

However, the use of liquid nitrogen for immediate comminution of the entire bulk

297

sample has been shown to provide considerably better results more efficiently. In

298

collaboration with the WESSLING lab, PROOF-ACS Germany assessed liquid nitrogen

299

comminution for the preparation of proficiency test samples for thiram,31 with which the

300

performance of 14 laboratories across Europe was evaluated. Organic strawberries (free of

301

incurred residues) were spiked with thiram at a level of 0.40 mg/kg, resulting in the formation

302

of the highly volatile CS2, which serves as the analyte. Another test material was prepared of

303

pears treated in the field with typical amounts of thiram and ziram before harvest. For the

304

first time in the production of proficiency test samples, degradation of dithiocarbamates was

305

negligible, which comes from using liquid nitrogen during comminution. Homogeneity

306

testing of 100 g subsamples of the bulk processed sample conducted by the WESSLING lab

307

gave a mean recovery of 95% of the spiking level with 5% RSD (n = 7).31

308

In the proficiency test study, both trueness and precision ( of the inter-lab results for

309

spiked strawberries were much better than previous studies, and the incurred pears also gave

310

more consistent results (trueness cannot be assessed without knowing the actual

311

concentration). When removing those lab results not meeting the z-score < |2| criterion, the

312

pear test sample was determined to contain 0.95 mg/kg thiram (as CS2) with 17% RSD (n =

313

10 labs), and the strawberry was measured to contain 0.34 mg/kg (85% recovery) with 15%

314

RSD (n = 10). In a previous proficiency test study using comminution at ambient

315

temperature, the recovery for thiram (determined as CS2) in lettuce was only 13% in spiked 13



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samples, and even the use of dry ice as a cooling agent led to a recovery of only 30%.32 The

317

regulatory acceptable (70-120%) recoveries obtained when quickly and easily using liquid

318

nitrogen demonstrates that it is a more efficient and effective option than traditional ambient

319

or cryogenic comminution with dry ice.

320

In summary, applying liquid nitrogen for sample processing in single-residue and

321

multiresidue pesticide analysis leads to superior results over conventional methods, including

322

cryogenic comminution with dry ice. Despite negative impressions of safety and other

323

practical issues, comminution with liquid nitrogen has been readily implemented in high-

324

throughput routine practice at low cost without difficulties, and in fact, the approach eases and

325

unifies sample processing procedures for difficult matrices analyzed by different methods.

326

Although homogeneity testing of < 1 g subsamples was not conducted in this study, we are

327

confident that the fine powders produced by this simple, one-step cryogenic procedure can

328

meet regulatory precision and trueness criteria needed for micro-samples to acceptably

329

represent the >500 g originally sampled bulk food commodities. We encourage others to

330

safely implement the advantageous liquid nitrogen comminution method as well, and report

331

the benefits provided, including gains in extraction efficiency, analyte stability, and

332

homogeneity testing of smaller test portions.

333 334

SAFETY NOTE: Although the WESSLING lab has been using liquid nitrogen routinely for

335

many years without incident, manufacturers of common comminution devices have not

336

designed nor tested their products specifically for their use in this application with liquid

337

nitrogen. Contact of liquid nitrogen with plastic components should be avoided.

338

REFERENCES 1. Lehotay, S. J; Cook, J. M. Sampling and sample processing in pesticide residue analysis. J. Agric. Food Chem. 2015, 63, 4395-4404. 2. Ambrus, Á.; Buczkó, J.; Hamow, K. Á.; Juhász, V.; Majzik, E. S.; Dobrik, H. S.; Szitás, R. Contribution of sample processing to variability and accuracy of the results 14


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of pesticide residue analysis in plant commodities. J. Agric. Food Chem. 2016, 64, 6071-6081. 3. Hajeb, P.; Herrmann, S. S.; Poulsen, M. E. Role of sample processing strategies at the European Union National Reference Laboratories (NRLs) concerning the analysis of pesticide residues. J Agric Food Chem. 2017, 65, 5759-5767. 4. Riter, L. S.; Wujcik, C. E. Novel two-stage fine milling enables high-throughput determination of glyphosate residues in raw agricultural commodities. J. AOAC Int. 2018, 101, 867-875. 5. Riter, L. S.; Lynn, K. J.; Wujcik, C. E.; Buchholz, L. M. Interlaboratory assessment of cryomilling sample preparation for residue analysis. J. Agric. Food Chem. 2015, 63, 4405-4408. 6. GOOD test portions: Guidance on obtaining defensible test portions. Association of American Feed Control Officials, Champaign, IL, USA, June 2018. 72 pp. www.aafco.org/Publications/GOODTestPortions 7. Han, L.; Lehotay, S. J.; Sapozhnikova, Y. Use of an efficient measurement uncertainty approach to compare room temperature and cryogenic sample processing in the analysis of chemical contaminants in foods. J. Agric. Food Chem. 2018, 66, 4986– 4996. 8. Lehotay, S. J.; Han L.; Sapozhnikova, Y. Use of a quality control approach to assess measurement uncertainty in the comparison of sample processing techniques in the analysis of pesticide residues in fruits and vegetables. Anal. Bioanal. Chem. 2018, 410, 5465–5479. 9. Lehotay, S. J.; Chen, Y. Hits and misses in research trends to monitor contaminants in foods. Anal. Bioanal. Chem. 2018, 410, 5331–5351. 10. Xu, G.; Hou, J.; Zhao, Y.; Bao, J.; Yang, M.; Fa, H.; Yang, Y.; Li, L.; Huo, D.; Hou, C. Dual-signal aptamer sensor based on polydopamine-gold nanoparticles and exonuclease I for ultrasensitive malathion detection. Sensors Actuators B: Chem. 2019, 287, 428-436. 11. Liu, M.; Wei, J.; Wang, Y.; Ouyang, H.; Fu, Z. Dopamine-functionalized upconversion nanoparticles as fluorescent sensors for organophosphorus pesticide analysis. Talanta 2019, 195, 706-712. 12. Fan, K.; Kang, W.; Qu, S.; Li, L.; Qu, B.; Lu, L. A label-free and enzyme-free fluorescent aptasensor for sensitive detection of acetamiprid based on AT-rich dsDNA-templated copper nanoparticles. Talanta 2019, 197, 645-652. 13. Liao, X.; Huang, Z.; Huang, K.; Qiu, M.; Chen, F.; Zhang, Y.; Wen, Y.; Chen, J. Highly sensitive detection of carbendazim and its electrochemical oxidation mechanism at a nanohybrid sensor. J. Electrochem. Soc. 2019, 166, B322-B327. 14. Zong, C.; Ge, M.; Pan, H.; Wang, J.; Nie, X.; Zhang, Q.; Zhao, W.; Liu, X.; Yu, Y. In situ synthesis of low-cost and large-scale flexible metal nanoparticle-polymer composite films as highly sensitive SERS substrates for surface trace analysis. RSC Adv. 2019, 9, 2857-2864. 15. Yaseen, T.; Pu, H.; Sun, D.-W. Fabrication of silver nanoparticles to simultaneously detect multi-class insecticide residues in peach with SERS technique. Talanta 2019, 196, 537-545. 16. Zeng, F.; Mou, T.; Zhang, C.; Huang, X.; Wang, B.; Ma, X.; Guo, J. Paper-based SERS analysis with smartphones as Raman spectral analyzers. Analyst 2019, 144, 137-142. 17. Wei, T.; Li, G.; Zhang, Z. A covalently cross-linked microporous polymer based micro-solid phase extraction for online analysis of trace pesticide residues in citrus fruits. J. Sep. Sci. 2019, 42, 888-896. 15



Page 16 of 18

18. Fussell, R. J.; Hetmanski, M. T.; Macarthur, R.; Findlay, D.; Smith, F.; Ambrus, Á.; Brodesser, P. J. Measurement uncertainty associated with sample processing of oranges and tomatoes for pesticide residue analysis. J. Agric. Food Chem. 2007, 55, 1062−1070. 19. Fussell, R. J.; Hetmanski, M. T.; Colyer, A.; Caldow, M.; Smith, F.; Findlay, D. Assessment of the stability of pesticides during the cryogenic processing of fruits and vegetables. Food Addit. Contam. 2007, 24, 1247-1256. 20. Gorji, S.; Biparva, P.; Bahram, M.; Nematzadeh, G. Rapid and direct microextraction of pesticide residues from rice and vegetable samples by supramolecular solvent in combination with chemometrical data processing. Food Anal. Methods 2019, 12, 394408. 21. Xue, J.; Zhang, D.; Wu, X.; Pan, D.; Hua, R. In-tube ultrasound assisted dispersive solid-liquid microextraction based on self-assembly and solidification of an alkanolbased floating organic droplet for determination of pyethroid insecticides in chrysanthemum. Chromatographia 2019, 82, 695-704. 22. Codex Alimentarius, Recommended methods of sampling for the determination of pesticide residues for compliance with MRLs, CAC/GL 33-1999. www.fao.org/faowho-codexalimentarius/thematic-areas/pesticides/en/ 23. Liang, D.; Liu, W.; Raza, R.; Bai, Y.; Liu, H. Applications of solid-phase microextraction with mass spectrometry in pesticide analysis. J. Sep. Sci. 2019, 42, 330-341. 24. Fussell, R. J. An overview of regulation and control of pesticide residues in food. ThermoFisher Scientific White Paper 71711-EN 0816M, 2016, 20 pp. 25. Guidance document on analytical quality control and method validation procedures for pesticide residues and analysis in food and feed. SANTE/11813/2017 26. Technical guideline on the evaluation of extraction efficiency of residue analytical methods. SANTE/10632/2017 Rev. 3 27. International Standards Organization. ISO/IEC 17025:2017 general requirements for the competence of testing and calibration laboratories. Mar. 2018 (corrected version). 30 pp. www.iso.org/standard/66912.html 28. Anastassiades, M.; Lehotay, S. J.; Stajnbaher, D.; Schenck, F. J. Fast and easy multiresidue method employing acetonitrile extraction/partitioning and dispersive solid-phase extraction for the determination of pesticide residues in produce. J. AOAC Int. 2003, 86, 412−431. 29. Community Reference Laboratory for Single Analyte Methods. Analysis of dithiocarbamate residues in foods of plant origin involving cleavage into carbon disulfide, partitioning into isooctane and determinative analysis by GC-ECD, Version 2. 2009. www.crlpesticides.eu/library/docs/srm/meth_DithiocarbamatesCs2_EurlSrm.PDF 30. Anastassiades, M. Pesticides which require special treatment during processing / homogenization and extraction. European Pesticides Residue Workshop, 2016, Limassol, Cyprus 31. Schindler, B.K. Method Ring Test P1510-MRT Dithiocarbamates in pears and strawberries, PROOF-ACS GmbH, Hamburg, Germany, 2015. www.proofacs.de/fileadmin/docs/P1510-RT_Dithiocarbamates_Summary.pdf 32. Lach & Bruns Consulting Chemists, Method performance assessment: Dithiocarbamates in lettuce purée samples. Hamburg, Germany. January, 2012

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Figure 1. Immediate comminution of fresh sample (strawberry) utilizing liquid nitrogen.

Figure 2. Comparison of traditional comminution (left) vs. use of liquid nitrogen (right) for difficult commodities: cocoa beans (top) and dried cranberries (bottom)

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Table 1. Degree of possible condensation on strawberry sample weight when applying liquid nitrogen (LN2) during comminution. The condensation shown on the outside of the container was the source of the weight increase after the initial 90 s.

Sample No.

Sample weight at 0 s (g)

Weight after 90 s using LN2 (g)

Weight increase after 90 s (%)

Weight after 180 s using LN2 (g)

Weight increase after 180 s (%)

1

642.4

649.1

1.04

642.7

0.05

2

667.3

678.1

1.62

667.8

0.07

3

650.2

653.5

0.51

650.4

0.03

4

666.8

680.0

1.98

667.1

0.04

5

650.2

656.6

0.98

650.4

0.03

18


Cryogenic Sample Processing with Liquid Nitrogen for

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