Article pubs.acs.org/JAFC

Development and Validation of a Standardized Method for the Determination of Morpholine Residues in Fruit Commodities by Liquid Chromatography−Mass Spectrometry Matt J. Hengel,*,† Rick Jordan,‡ and Wesley Maguire‡ †

Department of Environmental Toxicology, University of California at Davis, One Shields Avenue, Davis, California 95616, United States ‡ Pacific Agricultural Laboratory, 12505 N.W. Cornell Road, Portland, Oregon 97229, United States ABSTRACT: An analytical method was developed for the determination of morpholine on apples and citrus. The method utilized acidified methanol extraction, centrifugation, and determination by hydrophilic interaction liquid chromatography with electrospray ionization and tandem mass spectrometry (HILIC-ESI-MS/MS). Validation of the method occurred at the Pacific Agricultural Laboratory (PAL, Portland, OR, USA) and the Trace Analytical Laboratory (TAL, UC Davis, CA, USA). Method validation recoveries from control apple, orange, lemon, and grapefruit samples ranged from 84 to 120% over three levels of fortification (0.01, 0.04, and 0.2 μg/g). The limit of quantitation (LOQ) for all commodities was 0.01 μg/g, and the calculated method detection limit (MDL) ranged from 0.0010 to 0.0040 μg/g. KEYWORDS: fruits, liquid chromatography−mass spectrometry, HILIC-ESI-MS/MS, morpholine, residue method

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INTRODUCTION In the global market for fresh fruit, the United States is a major exporter of apples and citrus fruits to several countries. During the 2011−2012 growing season, 4.2 million metric tons of apples were produced with an estimated worth of U.S. $2.7 billion.1,2 Of this production, >20% was exported.2 In the case of citrus fruits, the 2011−2012 growing season yielded 10.6 million metric tons of citrus (orange, lemon, lime, tangerine, mandarin, and grapefruit) worth approximately U.S. $3.0 billion.3,4 Nearly 10% of the U.S. production of citrus was exported.4 Because a sizable portion of the domestic production is for export, growers need to be mindful of the import tolerances or maximum residue limits (MRLs) of pesticides and food additives for various countries. Apples and citrus fruits may be shipped over long distances, and the practice of applying thin wax coatings to protect the commodity has been in use since the 1930s.5 One component of the coating, morpholine, has come under scrutiny by the European Union (EU). Currently, morpholine is approved for use in the United States; however, morpholine has not been approved for use in the EU, and no MRL has been established.6,7 Although U.S. packing lines have been cleaned and morpholine-free waxes have been in place, inadvertent residues of morpholine at low levels can occur.8,9 In particular, morpholine has several other uses as a systemic fungicide, as an adhesive, as a fungicidal coating for paper products, and as a defoaming agent for pulp and paper production.10 Therefore, the possibility of dislodgeable residues from packaging materials may be of concern. Currently, there is no validated method in the United States to determine morpholine residues on waxed commodities. Therefore, the presented research on a rapid, selective, sensitive, and multilaboratory validated analytical method for the determination of morpholine on apples and citrus serves as a starting point for a domestically accepted method. © 2014 American Chemical Society

MATERIALS AND METHODS

Chemicals and Reagents. Morpholine (CAS Registry No. 110-91-8; 99.5%; Figure 1) was obtained from Chem Service Inc., West Chester,

Figure 1. Chemical structure of morpholine. PA, USA. All solvents were of pesticide grade or better. Reagents were of ACS grade or better. Water was prepared using a Milli-Q reagent water system. Specifications for filtration are cited below. Preparation of Standard Solutions. Stock solutions (1.00 mg/mL) of morpholine were prepared by adding 50 mg (corrected for purity) of the analytical grade compound to a 50 mL volumetric flask and bringing up to volume with acetonitrile (MeCN). Fortification solutions were typically prepared at 100, 10, 1.0, and 0.10 μg/mL by serial dilution in MeCN. The stock and fortification solutions were stored generally at ∼5 °C and were stable for 6 months. Calibration solutions for LC-MS/MS analysis were prepared by taking various volumes of the 0.10 μg/mL and higher concentrations of standard solutions and diluting them to volume in 20 mM ammonium formate in 60:40 water/MeCN (v/v, adjusted to pH 3.5 with formic acid) in the presence of clean crop extract (e.g., 200 μL of 0.1g/mL extract diluted to 1 mL with standard and solvent). Typically, calibration standards ranged from 0.50 to 50 pg/μL. Clean extracts from the uncontaminated control samples were prepared using the Special Issue: 50th North American Chemical Residue Workshop Received: November 26, 2013 Accepted: February 18, 2014 Published: February 18, 2014 3697

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extraction procedure detailed below. Calibration standards were prepared daily for each matrix type. Samples. Apple samples from the 2013 growing season, which either had no wax coating or had a morpholine-free coating, were provided by local growers. Whole citrus samples from the 2012 and 2013 growing seasons were provided by the USDA-IR4 program and had no coatings. All samples at the Trace Analytical Laboratory (TAL, UC Davis, CA, USA) facility were homogenized in the presence of dry ice using a Blixer food processor (Robot-Coupe USA, Inc.). Each chopped sample was stored in a labeled jar (approximately 1 L), and a lined lid was loosely closed on top to allow the dry ice to sublime during storage at −20 °C. All samples at the Pacific Agricultural Laboratory (PAL, Portland, OR, USA) facility were homogenized fresh using a Blendtec high-speed blender. Extraction. Fifteen grams of homogenized sample was weighed into a 50 mL polypropylene centrifuge tube, and water (1.4−2.4 mL) was added to yield 15 mL of total water based on moisture content of commodity.11 The addition of water aids in the extraction of morpholine. Recovery samples were fortified at this point, and 15 mL of 1% acetic acid in methanol was added to the tube. A ceramic homogenizer was added to each tube and shaken vigorously for 1 min by hand or using a mechanical shaker at 1000 strokes/min (Geno/ Grinder, model 2010, SPEX CertiPrep). Following extraction, the sample tubes were centrifuged at 4000 rpm for 5 min. The supernatant was poured off into clean culture tubes and stored at ∼5 °C prior to analysis. Typically, the extracts were stored for no more than 2 days. An aliquot of the extract was then diluted 1:5 with 20 mM ammonium formate in 60:40 water/MeCN (v/v, adjusted to pH 3.5 with formic acid) and filtered through a 0.2 μm PTFE syringe filter (VWR International) before it was submitted for LC-MS/MS analysis. Sample Analysis. At the PAL facility, sample analysis was conducted using a Waters Acquity UHPLC system (binary) coupled to a Waters Xevo tandem mass spectrometer via electrospray ionization (ESI). The TAL facility utilized an Agilent 1200 series binary LC coupled to an Agilent 6430 tandem mass spectrometer equipped with ESI. See Table 1 for instrument-specific conditions.

Table 2. Morpholine Residue Recoveries (Percent) in Apples and Citrus fortification levela commodity apple orange lemon grapefruit apple orange

0.01 μg/g

0.04 μg/g

0.2 μg/g

TAL Testing Facility 100 ± 2 107 ± 1 101 ± 2 107 ± 3 102 ± 1 107 ± 1 102 ± 2 109 ± 2 PAL Testing Facility 108 ± 13 104 ± 2 94 ± 4 92 ± 11 92 ± 2 92 ± 3

104 106 104 103

± ± ± ±

6 5 3 3

calcd MDLb (μg/g) 0.0018 0.0015 0.0010 0.0010 0.0040 0.0034

a

Replication for each fortification level, n = 7. bCalculated MDL: standard deviation at 0.01 μg/g fortification level × 3.143 (Student t99 (n − 1)).

Figure 2. Example standard curve of apple matrix-matched calibration standards from Agilent 6430.

Table 1. Mass Spectrometer Instrument Parameters parameter

Waters Xevo

Agilent 6430

source temperature desolvation temperature source gas flow (N2) cone/nebulizer gas flow (N2) capillary voltage collision gas morpholine primary transition morpholine secondary transition frag/collision energy (primary) frag/collision energy (secondary) ion ratio 42.1/70.1

150 °C 450 °C 800 L/h 5 L/h 1000 V Ar (0.11 mL/min) 88.1 → 70.1a 88.1 → 42.1b 30/12 30/12 20 ± 10%

300 °C N/A 11.5 L/min 50 psi 1000 V N2 (8 mTorr) 88.1 → 70.1 88.1 → 42.1 40/14 40/22 40 ± 10%

a Proposed collision-induced dissociation: [M + H]+ → loss of water (18). bProposed collision-induced dissociation: [M + H]+ → loss of HOCH2CH3 (46).

Chromatographic separation was accomplished with a SIELC Technologies hydrophilic interaction (HILIC) Primesep-A column (100 × 2.1 mm i.d., 5.0 μm particle size,). The PAL system utilized a column heater, which held the analytical column at 45 °C, whereas the TAL system was operated at ambient temperature (∼25 °C). The mobile phase was operated in isocratic mode with a composition of 20 mM ammonium formate in 60:40 water/MeCN (adjusted to pH 3.5 with formic acid) at 0.50 mL/min (TAL) and 0.55 mL/min (PAL). Following the completion of the analytical set, the column was flushed with 20 mM ammonium formate and then MeCN to remove accumulated matrix. Injection volume was 10 μL. Morpholine residues were quantified using a linear standard curve method with 1/x weighting. Typically, the primary transition was used for quantitation, and the secondary transition and ion ratio were used for compound confirmation.

Figure 3. Extracted ion chromatograms of 1 pg/μL (equivalent to 0.01 μg/g) calibration standard from Waters Xevo: (A) MRM transition 88 → 70; (B) MRM transition 88 → 42.

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RESULTS AND DISCUSSION Method Development. During the initial phases of method development, one of the main goals was to have a very simple and rapid sample workup that could provide a target limit of quanititation (LOQ) of 0.01 μg/g. This target 3698

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Figure 4. Extracted ion chromatograms of apple control sample fortified at 0.01 μg/g from Waters Xevo: (A) MRM transition 88 → 70; (B) MRM transition 88 → 42.

Figure 6. Extracted ion chromatograms of 1 pg/μL (equivalent to 0.01 μg/g) calibration standard from Agilent 6430: (A) MRM transition 88 → 70; (B) MRM transition 88 → 42.

to the analysis. An unpublished method by Anastassiades et al. described a procedure for very polar, non-QuEChERSamenable compounds in foods that utilizes acidified methanol extraction and determination by LC-MS/MS.14 This method met our needs in terms of simple and rapid sample workup but only showed recovery data on lime at 1.0 μg/g and extrapolated LOQs on apples and citrus at 0.01 μg/g.14,15 We therefore adopted a similar extraction procedure utilizing acidified methanol as in the Anastassiades method as a starting point for our method development. Initial attempts to run morpholine on a traditional reverse phase C18 column resulted in little to no retention and very poor peak shape under high-aqueous conditions. As morpholine has relatively high polarity, this observation was not unexpected. To improve retention and peak shape, a HILIC column was chosen. During early instrumental analysis fortified extracts were compared to external standards prepared in solvent to assess possible ion enhancement/suppression. It was

Figure 5. Extracted ion chromatograms of apple control sample from Waters Xevo: (A) MRM transition 88 → 70; (B) MRM transition 88 → 42.

was based on the default threshold which some governmental agencies, such as the EU, typically set at 0.01 μg/g on compounds that do not have established tolerances.12 Another goal was to have a method that utilized a relatively small amount of solvent and did not require the use of expensive and often difficult to obtain labeled internal standards. Gros et al. reported an LC-MS/MS based method for the determination of morpholine on pineapples which was able to achieve an LOQ of 0.01 μg/g.13 Although this method achieved the target LOQ, the procedure was not favorable as it required a dual solid phase extraction cartridge system, which adds additional time and cost 3699

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Figure 7. Extracted ion chromatograms of apple control sample fortified at 0.01 μg/g from Agilent 6430: (A) MRM transition 88 → 70; (B) MRM transition 88 → 42.

Figure 8. Extracted ion chromatograms of apple control sample from Agilent 6430: (A) MRM transition 88 → 70; (B) MRM transition 88 → 42.

observed that the response from the fortified extracts was suppressed >20% relative to the standard in solvent. As a result of these observations, the researchers chose to prepare matrixmatched standards for each crop type (apple and citrus) to correct the suppression effects. Method Validation. In addition to the goals above, the method needed to be rugged and transferrable to other laboratories that may utilize instrumentation from different vendors (e.g., Agilent or Waters). Therefore, method validation was conducted at both the PAL and TAL facilities. All control samples used for the validation studies showed no significant morpholine residues for apple or citrus above the instrument limit of detection (LOD) of 0.3 pg/μL as defined by a S/N > 3. The LOQ was defined as 1 pg/μL (0.01 μg/g equivalent), which achieved a S/N > 10. Recovery data (Table 2) generated at the PAL facility for apples and oranges ranged from 84 to 120% over three levels of fortification (0.2, 0.04, and 0.01 μg/g)

with all standard deviations ≤15% (n = 7 for each fortification level). The TAL facility produced recovery data for apples, oranges, lemons, and grapefruits that ranged from 97 to 112% over the same fortification levels listed above. As with the PAL data, good precision was observed at the TAL facility with all standard deviations ≤15% (n = 7 for each fortification level). Overall, the results from apple and orange method validation correlated very well between the two laboratories (Table 2). Lemons and grapefruits were also validated to assess the method on different types of citrus. As can been seen in Table 2, the results have excellent correlation to the results from the orange validation and suggest the method is suitable for citrus as a crop group. The coefficient of determination (R2) for morpholine was ≥0.99 at both facilities. An example calibration curve can be seen in Figure 2. Typical chromatograms of standards and crop extracts can be seen in Figures 3−8 for both instruments. Retention time differences can be 3700

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attributed to different flow rates, slight pH differences in the mobile phase preparation, and the usage of a column heater at the PAL facility. As a result of the research conducted, a rapid, rugged, and selective method was developed for the screening of morpholine in apple and citrus fruits. With the method presented herein, 50 samples can be extracted in ∼4 h and analyzed by LC-MS/MS overnight.

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(14) Anastassiades, M.; Kolberg, D. I.; Mack, D.; Sigalova, I.; Roux, D.; Fügel, D. Quick method for the analysis of residues of highly polar pesticides in foods of plant origins involving simultaneous extraction with methanol and LC-MS/MS determination (QuPPe-method). EU Reference Laboratories for Residues of Pesticides, version 6, 2011; http:// www.eurl-pesticides.eu (accessed Oct 2012). (15) Anastassiades, M.; Kolberg, D. I.; Mack, D.; Wildgrube, C.; Sigalova, I.; Roux, D.; Fügel, D. Quick method for the analysis of residues of highly polar pesticides in foods of plant origins involving simultaneous extraction with methanol and LC-MS/MS determination (QuPPe-method). EU Reference Laboratories for Residues of Pesticides, version 7.1, 2013; http://www.crl-pesticides.eu/library/docs/srm/ meth_QuPPe.pdf (accessed Nov 2013).

AUTHOR INFORMATION

Corresponding Author

*(M.J.H.) Phone: (530) 752-2402. Fax: (530) 754-8556. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Mike Willett from Northwest Horticultural Council for his valued support of this project.

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REFERENCES

(1) Noncitrus Fruits and Nuts 2011Summary. U.S. Department of Agriculture, National Agricultural Statistics Service, 2012; http:// usda01.library.cornell.edu/usda/nass/NoncFruiNu//2010s/2012/ NoncFruiNu-07-06-2012.pdf (accessed Sept 2013). (2) Fresh Deciduous Fruit (Apples, Grapes, & Pears): World Markets and Trade. U.S. Department of Agriculture, Foreign Agricultural Service, 2013; http://usda01.library.cornell.edu/usda/ current/decidwm/decidwm-06-21-2013.pdf (accessed Sept 2013). (3) Citrus Fruits 2013 Summary. U.S. Department of Agriculture, National Agricultural Statistics Service, 2013; http://usda01.library. cornell.edu/usda/current/CitrFrui/CitrFrui-09-19-2013.pdf (accessed Sept 2013). (4) Citrus: World Markets and Trade. U.S. Department of Agriculture, Foreign Agricultural Service, 2013; http://usda01.library. cornell.edu/usda/current/citruswm/citruswm-07-25-2013.pdf (accessed Sept 2013). (5) Baldwin, E. A. Edible Coatings for Fresh Fruits and Vegetables: Past, Present, And Future, Edible Coatings and Films to Improve Food Quality; Krochta, J. M., Baldwin, E. A., Nisperos Carriedo, M. O., Eds.; Technomic Publishing, Lancaster, PA, USA, 1994; p 25. (6) Code of Federal Regulation (CRF) Food and Drug Administration (FDA) 21CFR172.235; http://www.accessdata.fda. gov/scripts/cdrh/cfdocs/cfcfr/cfrsearch.cfm?fr=172.235 (accessed Feb 2014). (7) European Union Food Additives Database; https://webgate.ec. europa.eu/sanco_foods/main/index.cfm (accessed Sept 2013). (8) Willett, M. Northwest Horticultural Council, Yakima, WA, USA. Personal communication, 2013. (9) Good Fruit Grower; http://www.goodfruit.com/Good-FruitGrower/March-15th-2012/EU-regulations-stifle-fruit-exports/ (accessed Sept 2013). (10) National Organic Standards Board, Technical Advisory Panel Review of Morpholine, 2001; http://www.ams.usda.gov/AMSv1.0/ getfile?dDocName=STELPRDC5067002 (accessed Sept 2013). (11) Pesticide Analytical Manual, Vol. I: Multiresidue Methods, 3rd ed.; U.S. Food and Drug Administration: Washington, DC,, 1999; http:// www.fda.gov/downloads/Food/FoodScienceResearch/ucm111500. pdf (accessed Sept 2013). (12) European Union Pesticide Database; http://ec.europa.eu/ sanco_pesticides/public/index.cfm?event=commodity.selection (accessed May 2010). (13) Gros, P.; Matignon, F.; Plonevez, S. Determination of morpholine in fruits using LC-MS/MS. Ann. Falsif. Expert. Chim. Toxicol. 2011, 974, 17−22. 3701

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