Distribution of Animal Drugs among Curd, Whey, and Milk Protein

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Distribution of Animal Drugs among Curd, Whey, and Milk Protein Fractions in Spiked Skim Milk and Whey Nancy W. Shappell, Weilin L. Shelver, Sara J Lupton, Wendy Fanaselle, Jane M. Van Doren, and Heldur Hakk J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b04258 • Publication Date (Web): 04 Jan 2017 Downloaded from http://pubs.acs.org on January 8, 2017

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Journal of Agricultural and Food Chemistry

Distribution of Animal Drugs among Curd, Whey, and Milk Protein Fractions in Spiked Skim Milk and Whey

Nancy W. Shappell,†* Weilin L. Shelver,† Sara J. Lupton,† Wendy Fanaselle,‡ Jane M. Van Doren,‡ Heldur Hakk† †USDA-ARS, Biosciences Research Laboratory, 1605 Albrecht Blvd, Fargo, ND 58102-2765 ‡US-FDA, Center for Food Safety and Applied Nutrition, 5001 Campus Drive, College Park,

MD, 20740

AUTHOR INFORMATION *Corresponding Author: Email: [email protected] Phone: 701-239-1233 Fax: 701-239-1430

1 ACS Paragon Plus Environment


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ABSTRACT: It is important to understand the partitioning of drugs in processed milk and milk

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products, when drugs are present in raw milk, in order to estimate the potential consumer

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exposure. Radioisotopically labelled erythromycin, ivermectin, ketoprofen, oxytetracycline,

4

penicillin G, sulfadimethoxine, and thiabendazole were used to evaluate the distribution of

5

animal drugs among rennet curd, whey, and protein fractions from skim cow milk. Our previous

6

work reported the distribution of these same drugs between skim and fat fractions of milk. Drug

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distribution between curd and whey was significantly correlated (R2 = 0.70) to the drug’s

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lipophilicity (log P), with improved correlation using log D (R2 = 0.95). Distribution of drugs

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was concentration-independent over the range tested (20 – 2,000 nM). With the exception of

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thiabendazole and ivermectin, more drugs were associated with whey protein than casein on a

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nmol/mg protein basis (oxytetracycline experiment not performed). These results provide

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insights into the distribution of animal drug residues, if present in cow milk, among milk

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fractions, with possible extrapolation to milk products.

14 15 16 17 18 19

KEYWORDS: drug residues, curd, whey, skim milk, antibiotic, anthelmintic, NSAID, partition, distribution, protein association


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20 21 22 23

INTRODUCTION

Over the last decade, ≥ 99.96% of the more than 3 million annual U.S. bulk milk tanker

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samples were found to be free of violative drug residues.1 Historically, testing bulk milk

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deliveries of tanker milk to processing plants in the U.S. has been primarily focused on drug

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residues of beta-lactam antibiotics, which are commonly used in dairy cows when antibiotic

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treatment is required. However, other kinds of animal drugs are also administered to dairy cows

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when appropriate. Reports published in the National Milk Drug Residue Database by the U.S.

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Food and Drug Administration (FDA) confirm the presence of residues from drugs other than

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beta-lactam antibiotics in some samples from bulk tank or bulk milk pick-up tankers in the

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United States.2 The U.S. FDA published a risk-assessment entitled “Multicriteria-Based

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Ranking Model for Risk Management of Animal Drug Residues in Milk and Milk Products”,

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which used a science-based analytical approach to collate and incorporate relevant available data

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and information; and provided a decision-support tool to assist with reevaluating which animal

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drug residues should be included in milk testing programs.3 In preparing the risk assessment,

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data gaps, including the lack of drug residue distribution data in milk products, were identified. This

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information, describing partitioning of animal drugs in processed milk and its by-products, is

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necessary to ascertain the potential for human exposure. The complexity of assessing human

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exposures to drug residues from the products of one “liquid of biological origin” – cow milk – is

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evident when considering the multitude of products, including ice cream, yogurt, sour cream,

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various cheeses, whey protein supplements, and more than 35 others, derived from milk or

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whey.4 Understanding the factors driving the distribution and/or concentrating of animal drug



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residues among milk fractions will allow for better assessment of risk for potential human

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exposure.

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Literature describing distribution of drug residues during the processing of cow milk is

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limited, increasing somewhat when including studies of other species such as dairy goat, ewe,

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and bison.5-19 Most of the available studies have focused on antibiotics. Cerkvenik et al.8

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reported drug concentrations in milk products, such as yogurt or whey cheese, were both

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variable, and drug-dependent. Cayle et al.7 indicated that penicillin G (PENG) distribution was

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predominately in the aqueous phase with little distribution into fat-rich products such as cheeses.

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In two other penicillin related studies (other penicillins11, various antibiotics15), the physical and

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chemical properties of the specific compound affected drug distribution. The distributions of a

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number of parasiticides into curd and cheese, and the differential distributions in the milk

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products were dependent on the properties of the particular drug.5-7,16-17 Within a given drug

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class, the increase of residue concentrations in cheese relative to milk varied widely, from 1.5

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(albendazole10) to 13-fold (triclabendazole14). In both cases however, only metabolites were

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detected, not parent drug. In general, concern over residues in milk and milk products increases

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for drugs with long elimination half-lives, such as rafoxanide17, which persisted in milk for 47

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days, or ivermectin in buffalo or goat milk, with half-lives of approximately 2.5 days.5-6

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In our first paper we described “Phase 1” partitioning of animal drugs between the fat and

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skim fractions of pasteurized bovine whole milk, and created an empirical model that predicted

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drug distribution based on lipophilicity (logP).20 The model fit was improved by including the

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ionization state of the drug (logD). Seven drugs (Figure 1) that spanned four antibiotic classes

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[erythromycin (ERY), penicillin G, oxytetracycline (OTET), and sulfadimethoxine (SDMX)]

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two anthelmintics [ivermectin (IVR) and thiabendazole (THIA)]; and one analgesic [ketoprofen 4 ACS Paragon Plus Environment

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(KETO)] were studied. These results were the first reported measurements of this kind for many

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of these drugs. 20 In the present report, we measure the distributions of the same seven drugs

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between rennet skim milk curd (henceforth noted as “curd”) and whey fractions, casein proteins,

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and whey proteins using drug fortification and fractionation studies. The goal of this study was to

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better understand the factors that determine the distribution of animal drugs, if present in cow

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milk, among milk fractions, with possible extrapolation to milk products.

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MATERIALS AND METHODS

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Chemical, Supplies, and Equipment. Details pertaining to drugs, both unlabeled and

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radiolabeled, were reported previously. 20 Raw milk was obtained from the bulk milk tank at the

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North Dakota State University (NDSU, Fargo, ND) Dairy Unit farm (stored for ≤ 48 h after

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milking). Reference standards used to validate compositional analyses of various milk fractions

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(skim and whey) were obtained from Eurofins DQCI, (Mounds View, MN). Liquid vegetable

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rennet was obtained from New England Cheesemaking Supply Company (South Deerfield, MA).

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Ecolite® liquid scintillation cocktail was purchased from MP Biomedicals, LLC, Solon, OH.

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Carbo-sorb®, Permafluor®, and Monophase® were obtained from Perkin Elmer, Waltham, MA.

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Silica gel thin layer chromatography (TLC) plates (Uniplate™, 5 x 20 cm, 250 µm) and

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octadecyl (C-18) modified silica gel plates were purchased from Analtech, Inc. (Newark, DE).

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Amicon® Ultra-15 centrifugal filter (UF) devices were purchased from Millipore (Billerica,

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MA). Dremel tool and garlic press were standard retail products. A Precision® water bath

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(Thermo Scientific, Milford, MA), MagniWhirl® water bath (BlueM Electric Co., Blue Island,

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IL), and an Allegra X-14R centrifuge (Beckman-Coulter, Brea, CA) were used. A Tri-Carb 1900

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TR liquid scintillation counter (LSC, PerkinElmer, Houston, TX) and Model 307 Sample



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Oxidizer (Packard, Meridan, CT) were used for scintillation counting and sample oxidation,

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respectively. A Bioscan AR-2000 Imaging Scanner (Washington, DC) was used to detect

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radioactivity on TLC plates.

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Safety. Radiolabeled chemicals were handled in compliance with Nuclear Regulatory

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Commission (NRC) regulations for 14C and 3H.

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Determination of chemical purity and confirmation of chemical integrity. Thin layer

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chromatography analyses, and for some drugs, liquid chromatography tandem mass spectrometry

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(LC-MS/MS) were performed before and after the experiments. Initial analyses were used to

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evaluate dose purity, while post-incubation analyses were used to evaluate the extent of putative

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decomposition of the drug during processing. All TLC analyses were performed under

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conditions described in Hakk et al. 20

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Milk processing and radiochemical analysis.

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Drug Partitioning from Skim Milk into Curd and Whey Fractions (Phase 2). In previous work

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(Phase 1, Hakk et al.20) experiments were performed incubating the same set of radiolabeled

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drugs in whole milk followed by quantitation of drugs in the skim milk and fat fractions. Here, in

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Phase 2, drugs were incubated in skim milk prepared from whole pasteurized milk by

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centrifugation, as previously described. 20 Starting from fifteen tubes of whole milk (50 mL

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each), the skimmed fractions were combined into three pooled, replicate skim milk samples

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ensuring both randomness and homogeneity across the study. Partitioning of products was

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performed as diagramed in Figure 2 (Scheme 1). Skim milk (47 mL/tube) from pools 1, 2, and 3

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were fortified with 94 uL of 0, 10,000, 100,000, or 1,000,000 nM radiolabeled drug stock

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solutions to final concentrations of 0, 20, 200, and 2000 nM (three replicates per concentration).

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Due to the low specific activity of [14C] thiabendazole, 75 nM was utilized instead of 20 nM. 6 ACS Paragon Plus Environment

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One additional tube without spiked drug was prepared from each pool of skim milk to serve as

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matrix blanks (40 mL) for Kjeldahl analyses. Drug doses were prepared in water, acetonitrile, or

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methanol, depending on the drug solubility, with the final organic solvent concentration of the

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milk fraction never exceeding 0.2% (v/v). In order to determine possible non-matrix related

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analyte decomposition during equilibration and processing, duplicate water samples (20 mL

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each) were fortified with 200 nM drug (40 µL of 100 µM stock solution). After fortification, all

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tubes were vortexed for 5-10 sec, manually inverted and shaken to ensure complete mixing;

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aliquots were then removed for LSC (100 µL x 3) and TLC analyses. Tubes were then shaken at

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80 rpm for 30 min in a 38°C water bath (previously established as adequate for drug

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equilibrium). 20 A second sampling of the 2000 nM dose of skim milk (300 µL) was taken post-

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equilibration for TLC analysis.

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Curding was initiated by adding 172 µL of diluted rennet (~ 5% of stock vegetable rennet

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in nanopure water) to skim milk (~ 47 mL), vortexing, followed by a second incubation at 38° C

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(stationary water bath, 1 hour). Rennet was also added to water as a control, following the same

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procedure. Rennet curd was separated from whey by centrifugation (3,000 x g, 15 min, 20°C).

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Whey and water were sampled (3 x 200 µL) and assayed for radioactivity by LSC, and drug

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integrity assessed by TLC and/or LC-MS/MS. Curd was homogenized by passing twice through

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a hand-held garlic press, weighed into combustion cups (5 x 0.1 g aliquots per sample) and

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assayed for radioactivity by combustion and LSC.

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Determination of drug associated with whey proteins (Phase 3). Centrifugal ultrafiltration

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(10kDa molecular weight cut-off) was used to separate protein-associated drug from “non-whey

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protein-associated” (henceforth noted as “free”) drug. Fifteen tubes of pasteurized whole milk

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(50 mL each) and two tubes of nanopure water (20 mL each) were brought to 38°C in a 7 ACS Paragon Plus Environment


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stationary water bath, and whey was obtained as described above. Three separate pools of whey

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were created and distributed into tubes as described for skim milk preparations resulting in 15

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tubes of 20 mL each ( triplicate replicates, one replicate per pool for each dose level, and six

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blanks). Half the matrix blanks (0 nM) were used for determination of background radioactivity,

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total solids, and percent lipid and half were analyzed for protein using Kjeldahl as described in

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Hakk et al. 20 Whey was fortified using 40 µL of radiolabeled drug at 0, 10,000, 100,000, and

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1,000,000 nM to yield final concentrations of 0, 20, 200, and 2000 nM. Again, two water tubes

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were fortified to assess drug stability (200 nM). A 1 mL aliquot was removed from vortexed

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samples for LSC (100 µL x 3) and TLC analyses. Drug equilibration conditions for whey were

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the same as those used for skim milk (described above) and drug integrity was assessed post-

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equilibration using a 300 µL aliquot of each 2000 nM whey replicate. Post-equilibration, 15 mL

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of whey was transferred into an UF device, weighed, and centrifuged (4,000 x g, 17 min, 20°C)

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producing retentate (~5 mL) and permeate (~10 mL) fractions. Final fraction weights were taken

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prior to removal from UF device and density corrected to determine fraction volumes. The

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retentate was removed from the UF device, with ~ 1mL of retentate used to dislodge residual

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particulates on the filter, and added back to the retentate fraction prior to LSC. The filter was

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then washed with nanopure water (1 mL x 2) and the two washes combined prior to LSC. The

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washed retentate filters (two per UF device) were removed from the plastic housing using a

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Dremel tool and razor blade, air-dried overnight, weighed, placed in combustion cups, and

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oxidized in a sample oxidizer. The filter washes, permeate, and retentate were analyzed for

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radioactivity by LSC (200 µL x 3). TLC or LC-MS/MS analyses were performed to qualitatively

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assess drug integrity.


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Quantitation of drug-associated with casein and whey proteins. In Phase 3, drugs were

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incubated in whey, in the absence of the major milk proteins, caseins, which comprise ~ 84% of

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bovine milk proteins. 21 Therefore, additional experiments were performed to evaluate if drug

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affinity for whey proteins changed in the presence of caseins (Figure 3, Scheme 2). Twenty nM

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of drug (with exception of 75 nM for THIA) was added to 20 mL of skim milk and incubated for

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30 min. Post-incubation 15 mL of skim milk was processed through an UF device as described

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above for whey, enabling calculation of the percentage of drug associated with the total protein

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(casein + whey proteins) content of the UF skim milk retentate. In a second set of incubations,

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the drug was incubated with skim milk (20 nM in 47 mL), but was then processed with rennet to

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produce curd and whey. This whey (15 mL) was then subjected to the UF process. Drug

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remaining in the retentate in this case represented the proportion of drug that associated with

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whey proteins, while in the presence of casein.

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Differences in experimental conditions between Scheme 2, which is presented in Figure 3

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and Phase 3 above (see Scheme 1, Figure 2) were as follows: In Scheme 2, skim milk was spiked

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at only one drug concentration (20 nM). One matrix blank was included for each process instead

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of 3. The skim milk matrix required extending the ultrafiltration time to 2h in an attempt to

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obtain the v/v ratio of retentate/permeate previously obtained (~1:2) with Phase 3 whey filtration.

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In addition, more vigorous resuspension of retentate was required after the UF of skim milk,

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using a larger wash volume and three filter washes were performed. A fraction of the curd (5 x

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0.1 g) was combusted and counted to assess dose recovery. Filters were also combusted and

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counted as described above to determine non-specific binding.

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Compositional analyses. Compositional analyses were performed on the 0 nM blanks (no drug

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added, matrix blanks) of pasteurized skim milk, whey, curd, retentate, and permeate to yield 9 ACS Paragon Plus Environment


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percent lipid, total solids, and protein concentrations. For ultrafiltration experiments, aliquots

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from three matrix blank tubes were used for total solids and lipid composition, and aliquots from

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another three blanks were used for protein determinations. Method validation was confirmed by

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concomitant analysis of Eurofin standards. Procedures for total solids, fat, and protein

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determination were previously described.20 Briefly, volumes for total solids determinations were

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as follows: 1 mL for skim milk, whey, permeate and retentate, and 0.1 g for curd; for lipid

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content: 5 mL for each of skim milk, whey and permeate, and 1 mL retentate (diluted with 4 mL

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water), and 0.25 g curd (diluted with 5 mL water); for protein determination-total nitrogen: 5 mL

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for skim milk and permeate, 1 mL for retentate, and ~0.5 g for curd; for non-protein nitrogen: 10

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mL for skim milk, 2 mL for retentate; and for non-casein nitrogen: 10 mL for skim milk.

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Calculations for protein-associated drug (Figure 3). In the case of whey protein-associated

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drug, whey permeate after UF contained non-protein associated drug (‘free’ drug), while

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retentate contained both the protein associated drug and free drug. Permeate radioactivity and

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volume were used to calculate the dpms/mL so that the contribution from free drug could be

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subtracted from the radioactivity of the retentate. The radioactivity on the filter (obtained post-

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combustion) was treated as non-specific binding while the filter wash radioactivity was treated as

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part of the radioactivity of the retentate. The percent of whey protein-associated drug was

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calculated by dividing whey protein-associated radioactivity (‘bound’) by [dosed radioactivity

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minus non-specific binding radioactivity] and multiplying the result by 100. Similarly, the

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percent casein-associated drug was estimated by subtracting [percent drug associated with whey

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protein] from [percent drug associated with total N protein equivalents]. The percentage of whey

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protein-associated drug in the presence of casein was calculated based on the original spiked

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skim milk radioactivity so it was comparable to the total protein-associated drug. 10 ACS Paragon Plus Environment

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Statistical analyses

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Distribution data from all three incubation concentrations for curd/whey and retentate/permeate

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ratios (Phase 2 and 3, respectively) were analyzed for differences by ANOVA and linear

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regression. Retentate percent drug distribution was calculated relative to the total dpms (retentate

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+ permeate) and compared to retentate percent volume. A preferential association with whey

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protein was indicated by a higher percent of drug in the retentate than the percent of retentate

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volume, as analyzed by a paired two sample mean t-test.

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RESULTS AND DISCUSSION

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Compositional Analyses. Curding of skim milk yielded an average of 41 ± 0.4 mL of whey and

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5.1 ± 0.19 g of curd (COV ≤ 4%, data not shown). Skim milk and whey standards were used for

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QA/QC of composition analyses (Table S1). Laboratory values ranged from 97 to 102% of the

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reported standards for percent total solids, nitrogen, and true protein. However, laboratory values

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for percent lipid were consistently higher for the standards for both skim milk (0.16% versus

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0.09%), and whey samples (0.38% versus 0.28%, respectively). Balance inaccuracy was found to

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be the source of the overestimation when lipid weights were extremely low (< 0.3% of the milk

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fraction).

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Total solids, total N protein equivalent, true protein, and casein protein in skim milk

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prepared for incubation with drugs varied by ≤ 3%, and were ≥ to 96% of the values reported for

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skim milk used by Hakk et al.20 and ≥ 93% of the mean reported for standards (Table S1). Due

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to the low percentage of lipid in skim milk (≤ 0.3%) and consequent small lipid mass, the higher

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variability in lipid content was expected. Skim milk, curd, and whey composition were as

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follows: lipids (0.3, 2.0, and 0.2%); total solids (9, 31, and 7%); and total N (3, 24, and 1%, 11 ACS Paragon Plus Environment


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respectively). True protein was calculated from total N protein equivalents in skim milk, whey

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(whole or skim) and retentate, yielding 2.9, 0.7, and 2.3%, respectively. Casein protein in skim

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milk was 2.3%.

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Total N protein equivalents and true protein were determined on whey and its retentate

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fractions. Total N protein equivalents (0.19 ± 0.01%) in permeate fractions were equivalent to

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the non-protein N, confirming the integrity of the ultrafilters (Table S1). The increase in true

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protein in the retentate was proportional to the concentrating factor of ~ 3 (15 mL original

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volume to ~ 5 mL retentate volume). The starting whey was 0.91% total N and 0.68% true

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protein, while the respective values for retentate were 2.59% and 2.32%.

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Incubation period, drug stability, recovery, and dose response. Equilibrium conditions for all

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drugs in whole milk were achieved within 30 min when incubated at 37° C.20 Therefore drugs

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were allowed to equilibrate in skim milk for 30 min prior to rennet addition. Equilibration time

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of drugs in whey was evaluated using SDMX and KETO in triplicate incubations. Drug

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distribution was unchanged over 4 h (COV of mean % drug in retentate or permeate across 30

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min, 1, 2 and 4 h was ≤ 4%) and drug equilibrium was established in whey to be ≤ 30 min.

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Because equilibrium times of all drugs in whole milk were identical, the same was assumed for

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whey.

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Valid quantitation of drug distribution using radiolabeled drugs necessitates knowing that

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the drug stability was maintained throughout the experimental period. The integrity of 6 drugs

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was confirmed by radiochemical analysis of TLC plates at the following points: pre- and post-

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incubation in skim and whey, curd, and whey retentate fractions. The confirmation of OTET

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stability required LC-MS/MS analysis (data not shown) in addition to TLC. With the exception 12 ACS Paragon Plus Environment

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of KETO and PENG, drugs remained unchanged during milk partitioning. In 6 of 10 TLC

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analyses of KETO in curd, a shoulder appeared on the KETO peak, representing ~ 10-12% of the

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total activity (data not shown). In whey and retentate, radioactivity was evident in only one peak

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with the same Rf as cold KETO. The presence of lipids within the curd fraction might have

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interfered with the chromatographic behavior of KETO. The other drug exhibiting more than

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one peak was PENG, which is easily degraded.11 For PENG, broad peaks, peaks with shoulders,

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or split peaks were present in curd and whey. Reproducibility of data was evidenced by excellent

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dose recoveries with consistently small S.D.s (curd and whey experiments 98 ± 3.0%; and UF

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experiments 100 ± 2.2% across all doses and drugs, data not shown).

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The distributions observed across all drugs were generally independent of dose (SI Table

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2). While the ratios for both Phases 2 and 3 differed (P = 0.04) by dose for PENG, there was no

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dose dependence (P > 0.05). For SDMX, distribution was dependent on dose as assessed by both

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ANOVA (P = 0.002) and linear regression (P< 0.0002) in Phase 3, but the dose-dependent

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decrease in distribution ratio was 0.5%, essentially biologically irrelevant.

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Percent drug distribution from skim milk into whey and curd fractions (Phase 2). Prior to

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consideration of curd distribution data, one must remember that these data are specific to the

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methodology employed, and not interchangeable with processes that use lactic acid prior to renin

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addition in the curding process, as well as other variables such as pH. In addition, while cheese

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might be equated with curd protein, the fat content of cheese is highly variable, and therefore, the

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estimated drug distribution into cheeses would have to take into account the fat content and the

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lipophilicity of the specific drug.



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The range of drug distributing into the curd (including small amounts of whey) ranged

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from 14% (OTET) to 75% (IVR) (Figure 4). Reproducibility of data was evident with COVs for

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curd and whey distribution averaging 2% across doses (the highest COV of each 4% and 6%,

270

respectively). The percent of drug retained in curd appeared to be more strongly correlated with

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log D than MW. For example, the drugs with the largest relative distribution into curd, IVR

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(75%, log D 6.61, MW 875) and THIA (48%, log D 2.93, MW 201) had the highest log D values

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among the drugs examined. For drugs with intermediate log D values, like SDMX (1.23) and

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ERY (1.24), curd retained ~ 30% of the drug; while for drugs with a negative log D (OTET,

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PENG, and KETO) ~ 13% of drug was entrained in curd.

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Few reported data are directly comparable to our findings, as drugs were typically

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administered to animals, and incorporation into milk and milk products (predominately cheese)

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evaluated. One exception was a report by Cayle et al.7, evaluating whole milk fortified with

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PENG, in which the same percentage of PENG (12%) was found in cheese (42% moisture) as

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was observed with fortified skim milk in this study (Table S4, 12 ± 0.3%). One abstract reported

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that chlortetracycline (structurally similar to OTET) was initially at the highest concentration in

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the whey fraction post-mammary infusion of the drug (as detected by microbiological

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assessment), yet in later post-infusion milkings, concentrations were higher in the casein

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fraction. 22 Initial concentrations post-infusion may not have reflected equilibrium with milk

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proteins in the gland cistern, and later distributions may have been the result of drug absorption

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by the gland and de novo incorporation into milk. While we found the majority of OTET in the

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whey fraction, the concentration was actually higher for OTET in the curd or casein fraction.


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When sheep were dosed subcutaneously with IVR, an average of 57% of the drug from

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whole milk was retained in cheese (extrapolated moisture content of 48%) compared to 75%

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measured in the present study after spiking skim milk.8 Cerkvenik et al8 reported a mean of 9%

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for IVR in whey, versus this study’s 21 ± 0.7% in spiked skim milk (Table S4). A partial

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explanation for the differences in % IVR found in whey might be differences in recovery (this

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study a  = 95 ± 2.1% across all doses, 65%).8 Another factor may be differences in the amount

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of fat. Others reported 66% of IVR from whole sheep’s milk partitioned into cheese, with 38%

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associated with whey.12

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Thiabendazole (THIA) results from this study are similar to findings with albendazole, a

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drug with a similar structure. Fletouris et al.10 orally dosed cows with albendazole, and reported

298

that 30% of the albendazole initially present in whole milk (as metabolites) was associated with

299

curd and 70% was associated with whey. In the current study using skim milk, 47% of THIA

300

associated with curd and 54% with whey (Table S4). Using Phase 120 and Phase 2 data, the %

301

THIA associated with curd and whey derived from whole milk would be ~ 37 and 40%,

302

respectively, closer to the findings of Fletouris et al10. Again, direct comparison is problematic

303

when evaluating drug distribution when starting with whole versus skim milk, especially for

304

highly lipophilic drugs such as IVR.

305

The curd produced in our experiments contained small amounts of whey. To adjust for

306

this, the moisture content of curd was assumed to have retained the same concentration of whey

307

protein as that of whey, and the value for drug concentration in whey was multiplied by the curd

308

liquid weight and subtracted from the drug concentration of curd. This value is referred to as

309

“0% moisture curd”. The difference in percentage of drug residue in “experimental” versus “0%



Page 16 of 41

310

moisture” curd is presented in Table S3. Accounting for whey in the curd reduced the

311

percentage of curd-associated drug by 30 to 60% for all drugs but THIA and IVR, which

312

changed by less than 10%.

313

Drug concentration in curd relative to milk (Phase 2). Figure 4 shows that drug concentration

314

increases in the curd relative to the initial skim milk concentration in every case, with the

315

smallest increase found with PENG (110% of skim milk) and the largest at more than 500%

316

(IVR). Since the mass of curd produced from the starting skim milk is ~10% of the whey

317

produced, an increase in drug concentration could be expected, even without preferential affinity

318

for curd proteins. Consistent with our results, Adetunji et al.23found essentially no change in

319

PENG concentration of curd and cheese relative to whole milk concentrations at 3 of 5 milk

320

processors. Data from the same study using streptomycin and tetracycline found inconsistencies

321

across processors, with curd and cheese concentrations sometimes lower than the original raw

322

milk. This is in contrast with our findings for OTET, where a 40% increase in concentration was

323

found in the curd. It is unclear why the lack of consistency was present across processors, but it

324

may reflect differential methodologies, along with potential for more degradation of the

325

antibiotics during milk processing.

326

Literature reports of anthelmintic in curd and cheese were somewhat similar to those

327

reported here. IVR in cheese from sheep and/or buffalo whole milk had concentrations from

328

290% 12to 400% 13,6 those of the original milk. Another anthelmintic of similar structure,

329

eprinomectin (MW 914), was found in curd at 375% of the concentration of the whole sheep’s

330

milk.13 The concentration in curd relative to skim milk was intermediate for THIA (410% of


Page 17 of 41


331

skim, this study), in contrast to the 1300% in cheese produced from whole sheep’s milk for

332

another structurally similar drug, triclabendazole (based on metabolites).14

333

Empirical modelling of drug distribution between curd and whey fractions. The log

334

concentrations in curd to whey versus the log P or D value (pH 6.8) are presented in Figure 5,

335

Panels A and B; numerical values are reported in SI Table 3. While both log P and D were

336

correlated with the distribution ratio of drugs in curd and whey, the impact of ionizable groups

337

accounted for by the log D value resulted in a stronger linear correlation (R2 = 0.95 versus 0.70

338

for log P). Specifically, the fit improved for the four drugs with ionizable groups (PENG, KETO,

339

THIA and IVR; see SI for pKa’s) resulting in charged molecules at the pH range of milk

340

fractions examined (pH 6.6 - 6.8). Similar results were obtained for a model of drug distribution

341

between fat and skim milk.20 Figure 5 (Panels C and D) also show drug distribution ratios for

342

0% moisture curd and whey; while changes in slope and intercept for both log P and log D

343

graphs occurred, the R2 values remain essentially unchanged. Reporting drug distribution on a

344

0% moisture curd basis would allow for modeling of concentrations in curd with variable

345

moisture content.

346

When considering the distribution of incurred animal drug residues in dairy products, it is

347

essential to describe the distribution among all of the components in the original whole milk.

348

Figure 6 combines the results of the present study with those of Phase 120 to provide drug

349

partitioning relative to whole milk. For example, while IVR concentration in curd was ~33 times

350

higher than in whey (and ~7 times the original concentration from an incubation of skim milk), ~

351

87% of IVR would be removed with lipid during skimming, resulting in only ~13% of original

352

concentration for distribution between the skim milk rennet curd (~10%) and whey (~3%).



Page 18 of 41

353

Drug-association with whey protein as determined by centrifugal ultrafiltration – retentate

354

versus permeate (Phase 3). Whey produced from whole milk was fortified with radiolabeled

355

drug, and ultrafiltered. The distribution of drug between retentate and permeate fractions are

356

shown in Figure 7. Assuming no drug-protein association, drug distribution in whey would

357

simply reflect the volume distribution of retentate and permeate (Figure 7 solid lines). The

358

distributions for all drugs were significantly different (P

435

80% of the milk proteins, and are preferentially coagulated in the curding process. By contrast,

436

in a pure casein solution in PBS of 2.66 mg/ mL, binding was reported as only 8%,11somewhat

437

smaller than our findings of 16% (Table 1). The difference may be due to experimental

438

conditions, where pure casein was dialyzed at 4° C for several days in the study by Grunwald

439

and Petz11, with the potential for degradation of PENG during dialysis. In addition, casein may

440

have become saturated since their concentration was 1/10 that of normal cow’s milk (~28 mg/ 21 ACS Paragon Plus Environment


Page 22 of 41

441

mL). In contrast, in vivo administration of PENG to cattle resulted in 28% and 33% of PENG in

442

serum to be protein-associated (based on dialysis or ultrafiltration separation, and assay of

443

radioactivity or antibiotic activity, respectively). Interestingly, these results for bovine blood

444

proteins are similar to the present findings of 16% for casein, but lower than those reported by

445

Drug Bank25 a data base of over 8,000 drugs providing chemical, pharmacological and

446

pharmaceutical information (45 to 68%, protein unspecified). These data demonstrated that

447

PENG binding can differ widely depending on the sources of proteins.

448

No data has been published on OTET association with milk proteins, but Ziv and

449

Rasmussen17 did report the distribution of the related compound, tetracycline, in milk from goats

450

receiving the drug in vivo. Their finding that 52% of tetracycline was associated with milk

451

proteins in skim milk is much higher than reported here for curd (7%) or whey associated protein

452

(8%). The structure, MW, log P, and log D (-2.50 and -2.93 calculated at pH 6.8) values for

453

OTET and tetracycline are very similar, providing no rationale for the differences observed. But,

454

differences for protein association among OTET, tetracycline, and chlortetracycline were

455

observed by Ziv and Sulman24, when the drugs were administered in vivo to either cows or ewes.

456

The percent associated with serum was lowest for OTET (~20%), followed by tetracycline (~

457

35%), and then chlortetracycline (~48%). The values reported by the Drug Bank25 ranged from

458

20% to 67% for tetracycline.

459

While there are no literature reports for the distribution of ERY in milk, 13% of

460

spiramycin (a structurally similar antibiotic) was associated with skim milk proteins after in vivo

461

administration to goats.19 This finding is similar to the 12 and 15% found for curd protein and

462

whey protein, respectively, in the present study. Literature reports for binding to bovine serum


Page 23 of 41


463

protein were lower: 18% by dialysis, and 20% by UF23, although the Drug Bank25 reported very

464

high association values (75-95%) possibly due to different sources of protein or experimental

465

methods. Drug Bank25 values for the rest of the drugs were consistently higher than either whey or

466 467

casein protein, even the calculated casein association. Two antibiotics, sulfamethoxazole and

468

sulfapyridine, chemically similar to SDMX, were reported by the Drug Bank25 to bind 50% and

469

70% of unspecified protein, respectively, while the present study reported 37% of SDMX to be

470

casein-associated. The sum of curd and whey protein associated THIA was 38% in our study. To

471

the best of our knowledge, association of THIA with milk proteins has not been reported

472

previously.

473

In conclusion, this paper provides an empirical model for predicting animal drug

474

distribution between rennet skim milk curd and whey. Results from this study also characterize

475

drug residue protein-associations. However, caveats must be considered that include the in vivo

476

distribution of drugs may differ from in vitro laboratory equilibrations, specifics of industry

477

processing which may result in different distributions, and metabolites or degradation products,

478

when present, which may distribute differently. If a drug is present in cow milk, the data

479

reported here and in our first paper20 presents a quantitative model for use as a food safety tool in

480

estimating drug distribution and concentration among milk fractions for various types of animal

481

drugs.

482

ABBREVIATIONS

483

ERY, erythromycin; IVR, ivermectin; KETO, ketoprofen; OTET, oxytetracycline; PENG,

484

penicillin G; SDMX, sulfadimethoxine; and THIA, thiabendazole.



Page 24 of 41

485

ACKNOWLEDGMENTS

486

The authors wish to acknowledge the helpful discussions provided by Dennis Gaalswyk, Yinqing

487

Ma, David Oryang, and Chi Yuen Yeung from the FDA. We wish to thank the technical

488

assistance provided by Dee Ellig, Patrick Harland, Lindsey Fransen, Amy McGarvey, Jason

489

Neumann, Colleen Pfaff, and Michael Woodworth from the ARS Biosciences Research

490

Laboratory. Appreciation also goes to Todd Molden and Thomas Brown for milk collection

491

from the NDSU dairy barn.

492

SUPPORTING INFORMATION

493

Compositional analysis of skim milk, milk fractions, and reference standards; statistical analysis of dose

494

responses; log D and P values used for regression analyses of drugs; and data summaries for individual

495

drugs for Phase 2 and 3 (Tables S5-S18) are included in the supporting information.

496

FUNDING

497

This study was collaboratively funded by an interagency agreement with the FDA and USDA

498

ARS (Interagency Agreement no. 224-14-2006).

499

NOTES

500

The use of trade, firm, or corporation names in this publication is for the information and

501

convenience of the reader. Such use does not constitute an official endorsement or approval by

502

the United States Department of Agriculture (USDA), the Agricultural Research Service, or the

503

Food and Drug Administration of any product or service to the exclusion of others that may be

504

suitable. USDA and FDA are each equal opportunity employers.

505

The authors declare no competing financial interest.

506 507


Page 25 of 41


508

REFERENCES

509

(1) National Milk Drug Residue Database, FY 2005 - FY2015, www.kandc-sbcc.com/nmdrd/.

510

(accessed November 21, 2016).

511

(2) Food and Drug Administration. Milk drug residue sampling survey.

512

http://www.fda.gov/downloads/AnimalVeterinary/GuidanceComplianceEnforcement/Complianc

513

eEnforcement/UCM435759.pdf, 2015 (accessed August 11, 2016).

514 515 516 517

(3) Food and Drug Administration. Multicriteria-based ranking model for risk management of animal drug residues in milk and milk products. http://www.fda.gov/Food/FoodScienceResearch/RiskSafetyAssessment/ucm443549.htm, 2015 (accessed September 4, 2015).AAAA

518

(4) Smith, K. Dried dairy ingredients. Wisconsin Center for Dairy Research. 2008.

519 520 521

(5) Anastasio, A.; Esposito, M.; Amorena, M.; Catellani, P.; Serpe, S.; Cortesi, C. Residue study of Ivermectin in plasma, milk, and mozzarella cheese following subcutaneous administration to buffalo (Bubalus bubalis). J. Agric. Food Chem. 2002, 50, 5241-5245.

522 523 524

(6) Anastasio, A.; Veneziano, V.; Capurro, E.; Rinaldi, L.; Cortesi, M.; Rubino, R.; Danaher, M.; Cringoli. G. Fate of Eprinomectin in goat milk and cheeses with different ripening times following pout-on administration. J. Food Prot. 2005, 68, 1097-1101.

525 526

(7) Cayle, T.; Guth, J. H.; Hynes, J. T.; Kolen, E. P.; Stern, M. L. Penicillin distribution during cheese manufacture and membrane treatment of whey. J. Food Prot. 1986, 49,796-798.

527 528 529

(8) Cerkvenik, V.; Perko, B.; Rogelj, I.; Doganoc, D. Z.; Skubic, V.; Beek, W. M. J.; Keukens, H. J. Fate of ivermectin residues in ewes’ milk and derived products. J. Dairy Res. 2004, 71, 3945.

530 531 532

(9) De Liguoro, M.; Longo, F.; Brambilla, G.; Cinquina, A.; Bocca, A.; Lucisano, A. Distribution of the anthelmintic drug albendazole and its major metabolites in ovine milk and milk products after a single oral dose. J. Dairy Res. 1996, 63, 533-542.

533 534 535

(10) Fletouris, D. J; Botsoglou, N. A.; Psomas, I. E.; Mantis, A. I. Albendazole-related drug residues in milk and their fate during cheesemaking, ripening, and storage. J. Food Prot. 1998, 61, 1484-1488.

536 537

(11) Grunwald, L.; Petz, M. Food processing effects on residues: penicillins in milk and yoghurt. Anal. Chim. Acta. 2003, 483, 73-79.



Page 26 of 41

538 539 540

(12) Imperiale, F.A.; Busetti, M. R.; Suarez, V. H.; Lanusse, C. E. Milk excretion of ivermectin and moxidectin in dairy sheep: assessment of drug residues during cheese elaboration and ripening period. J. Agric. Food Chem. 2004, 52, 6205-6211.

541 542 543

(13) Imperiale, F. A.; Pis, A.; Sallovitz, J.; Lifschitz, A.; Busetti, M.; Suárez, V.; Lanusse, C. Pattern of eprinomectin milk excretion in dairy sheep unaffected by lactation stage: Comparative residual profiles in dairy products. J. Food Prot. 2006, 69, 2424-2429.

544 545 546

(14) Imperiale, F.; Ortoz, P.; Cabrera, M; Farias, C.; Sallovitz, J. M.; Lezzi, S.; Perez, J.; Alvarez, L.; Lanusse, C. Residual concentrations of the flukicidal compound triclabendazole in dairy cows’ milk and cheese. Food Addit. Contam. 2011, 28, 438-445.

547 548 549

(15) Iezzi, S.; Lifschitz, A.; Sallovitz, J.; Nejamkin, P.; Lloberas, M.; Manazza, J.; Lanusse, C.; Imperiale, F. Closantel plasma and milk disposition in dairy goats: assessment of drug residues in cheese and ricotta. J. Vet. Pharmacol. Therap. 2014, 37, 589-594.

550 551 552 553

(16) Power, C.; Sayers, R.; O’Brien, B.; Clancy, C.; A. Furey, A.; Jordan, K.; Danaher, M. Investigation of the persistence of closantel residues in bovine milk following lactating-cow and dry-cow treatments and its migration into dairy products. J. Agric. Food Chem. 2013a, 61, 87038710.

554 555 556

(17) Power, C.; Danaher, M.; Sayers, R.; O’Brien, B.; Whelan, M.; Furey, A.; Jordan, K. Investigation of the persistence of rofoxanide residues in bovine milk and fate during processing. Food Addit. Contam. 2013b, 30, 1087-1095.

557 558 559

(18) Whelan, M.; Chirollo, C.; Furey, A.; Cortesi, M. L.; Anastasio, A.; Danaher, M. Investigation of the persistence of levamisole and oxyclozanide in milk and fate in cheese. J. Agric. Food Chem. 2010, 58, 12204-12209.

560 561

(19) Ziv, G.; Rasmussen, F. Distribution of labeled antibiotics in different components of milk following intramammary and intramuscular administration. J. Dairy Sci. 1975, 58, 938-946.

562 563 564 565

(20) Hakk, H.; Shappell, N. W.; Lupton, S. J.; Shelver, W. L.; Fanaselle, W.; Oryang, D.; Yeung, C. Y.; Hoelzer, K.; Ma, Y.; Gaalswyk, D.; Pouillot, R.; Van Doren, J. M. Distribution of Animal Drugs between Skim Milk and Milk Fat Fractions in Spiked Whole Milk: Understanding the Potential Impact on Commercial Milk Products. J. Agric. Food Chem. 2016, 64, 326-335.

566 567

(21) Anderson, R.R.; Collier, R.; Guidry, A.; Heald, C.; Jenness, R.; Larson, B.; Tucker, H.A. Lactation. Chapter 5, p 178, 190-191. Iowa State University Press, Ames, IA. 1985.

568 569

(22) Rusoff, L.; Lee, G.C.; Stone, E. J. Aureomycin (Chlortetracycline) distribution in milk. J. Dairy Sci. 1957, 40:1390.

570 571

(23) Adetunji, V. O. Effects of processing on antibiotic residues (streptomycin, penicillin-G and tetracycline) in soft cheese and yoghurt processing lines. Pakistan J. Nutri. 2011, 10, 792-795.


Page 27 of 41


572 573

(24) Ziv, G.; Sulman, F.G. Binding of antibiotics to bovine serum. Antimicrob. Agents Chemother. 1972, 2, 206-213.

574

(25) Drug Bank Database. http://www.drugbank.ca/ (accessed March 18, 2015).



575 576 577 578 579 580 581 582 583 584

Figure Legend

585 586 587 588 589 590 591 592 593 594 595 596 597 598 599 600 601 602 603

Figure 3. Scheme (2) of milk partitioning for determination of percent drug associated with whey or casein protein. Results indicated in boxes, OTET not assayed, as protein associated fraction too small to accurately quantify.

Page 28 of 41

Figure 1. Structures of animal drugs used in the milk partitioning studies. a) Radioactively labeled with a single, general tritium atom, i.e. location of label is unknown. b) Asterisk indicates site of radiolabel. Asterisk within an aromatic ring indicates the ring was uniformly labeled. Figure 2. Scheme (1) of milk partitioning processes that yielded curd and whey from skim milk (Phase 2) and retentate and permeate from whey (Phase 3).

Figure 4. Drug distribution and relative concentration ratios from skim milk into whey and curd fractions. Bars represent percent mean of all concentrations (n=3 concentrations; n=3 replicates per concentration) ± standard deviation of all three dose mean percentages based on disintegrations per minute (dpm) of whey and curd (at 70% moisture) fractions compared to fortified skim milk dpm. Numerical values on graph represent the mean ratio (n=3) of the drug concentration in the fraction (curd or whey) to the initial drug concentration in skim milk ± SD. Sum of stacked plots represents total, unadjusted drug recovery values. Figure 5. Regression analyses of observed log [Drug]curd or 0% moisture curd/[Drug]whey (log C/W or 0%mC/W) vs. log P and log D (pH 6.8) using natural y intercept. Graph A is the regression analysis of log C/W vs. log P. Graph B is the regression of log C/W vs. log D (pH 6.8). Graph C is the regression analysis of log 0%mC/W vs. log P. Graph D is the regression of log 0%mC/W vs. log D (pH 6.8). Drug Bank pKas accessed on 2-11-2015 (www.drugbank.ca). Log P accessed from Chemspider on 1-28-2015 (www.chemspider.com). Calculations were performed as: log Dacid = log P + log[1/(1+10pH-pKa)] or log Dbase = log P + log[1/(1+10pKa-pH)].

604 605 606 607 608 609

Figure 6. Normalized percentages of animal drugs calculated to be in the milk end products (a) milk fat, (b) curd, (c) permeate, and (d) retentate based on data generated from the current studies. Percentage values in the curd and retentate bars represent pure curd percent and drug-towhey protein associations normalized to whey percentages. SDMX bar has additional information on which milk end products comprise whole milk, skim milk, high-fat curd, low-fat curd, and whey, as a guide to where drug may partition during commercial milk processing.

610 611 612 613 614 615

Figure 7. Drug distribution and relative concentration ratios of retentate and permeate fractions produced from fortified whey originating from whole milk. Bars represent percent mean of all concentrations (n=3 concentrations; n=3 replicates per concentration) ± standard deviation of the three dose means based on disintegrations per minute (dpm) of retentate and permeate fractions normalized to the total dpm between retentate and permeate. Numerical values on graph represent the mean ratios (n=3) of the drug concentration in the fraction (retentate or permeate) 28 ACS Paragon Plus Environment

Page 29 of 41


616 617 618

to the initial drug concentration in whey ± SD. Horizontal lines represent retentate and permeate percentage volumes from the filtration process. All drug distribution percentages were statistically different than volume percentages, p < 0.05.

619 620 621 622 623 624 625 626 627 628 629

Figure 8. Milk partitioning scheme for all seven animal drugs, including the radiochemically assayed partitioning values, for all fractions, i.e. Phases 1 to 3. Whey and curd for Phase 2 were produced from fortified skim milk. In Phase 3, whey was produced from whole milk, then fortified and retentate and permeate fractions prepared by ultrafiltration. The wheys produced from skim milk and whole milk were similar as seen in the compositional data from Table S1. Milk fat and skim partitioning values (Phase 1) are from Hakk et al. 2016. The moisture content of curd was assumed to reflect drug associated with whey, and was subtracted out to obtain the “0 % Moisture Curd” value. The % of drug that was whey protein-associated was calculated by difference (total in retentate minus concentration in permeate times the concentrating factor of retentate).



Page 30 of 41

630

Table 1. Percentages of the extent that seven animal drugs in the current study associated with various milk protein fractions as described in Figure 3. Comparisons are also made to available literature values, footnotes with citation and differences in methodology. This Study, Casein Associated Drug

This Study, Whey Protein Associated

PENG

16

OTETd

24

25

Literature, Milk Proteins

Literature , Bovine Seruma (D-Dialysis; UFUltrafiltrate)

Drug Bank Unspecified Protein

7

5 (whole milk)b,11 9 (skim milk)c,19 8 (pure casein)b,11

28 (D), 33(UF)

45-68

7

8

52 (skim milk)e,19

20-67g

ERY

31

11

13 (skim milk)f,19

19(D), 22 (UF) 18(D), 20 (UF)

SDMX

37

8

KETO

38

17%

99

THIA

48

7%

70h

IVR

77

21%

93

Drug

75-95 50, 70g

a

in vivo administration of drug to cows; bovine serum, UF 1:10 retentate:permeate, dialysis 1 part of serum into 2 parts of buffer b bench-top fortification of milk or casein with drug c in vivo administration to goats d In this study, protein association too low for quantitation of OTET, values presented are from Phase 2 (curd) and 3 (whey protein) determined associations. e in vivo administration to goats, similar drug: tetracycline f in vivo administration to goats, similar drug: spiramycin g similar drugs: Sulfamethoxazole (70%) sulfapyridine (50%) h similar drug: albendazole


Page 31 of 41


Table 2. Calculated protein associated drug (nmoles per gram of casein or whey protein). Whey Whey Casein Casein Casein Protein Protein nmol/g Assoc./ Assoc./ [Incubation] nmol/g nmol/g (Phase 2 0% Drug Whey Whey nM (Phase 3 (Scheme 2 Moisture Assoc. (w/o Assoc. (w/ w/o w/ Curd)a casein) casein) casein) casein)b 20 0.07 0.24 nac 0.3 OTET 200 0.77 2.52 na 0.3 2,000 7.97 23.3 na 0.3 20 200 2,000

0.04 0.44 4.10

0.18 1.98 19.7

0.18

0.2 0.2 0.2

0.2

PENG

20 200 2,000

0.14 1.41 14.5

0.44 4.12 43.7

0.28

0.3 0.3 0.3

0.5

ERY

0.20 1.87 18.6

0.44 4.48 37.7

0.26

0.5 0.4 0.5

0.8

SDMX

20 200 2,000

0.19 1.86 18.2

0.74 6.91 84.5

0.46

0.3 0.3 0.2

0.4

KETO

20 200 2,000 75 200 2,000

1.50 3.82 39.6

1.02 2.90 31.1

0.61

1.5 1.3 1.3

2.5

THIA

20 1.08 3.25 0.55 0.3 2.0 200 8.32 31.2 0.3 2,000 88.4 308 0.3 a These data have whey associated drug subtracted, and were calculated for “0% moisture curd” consisting of casein protein (see text for details). b These data were obtained when drug was incubated with skim milk, curded, then the whey fraction subjected to centrifugal ultrafiltration to determine drug associated with whey proteins. These values would be more reflective of incurred drug distribution. Scheme 2 was not done with OTET, as counts were too low to reliably quantitate on a protein basis. c na is not analyzed. IVR

631



Page 32 of 41

632

Figure 1. Structures of animal drugs used in the milk partitioning studies. a) Radioactively labeled with a single, general tritium atom, i.e. location of label is unknown. b) Asterisk indicates site of radiolabel. Asterisk within an aromatic ring indicates the ring was uniformly labeled.

* * 3

a

H(G)-Oxytetracycline (OTET) Tetracycline Antibiotic

14

3

14

b

C-Sulfadimethoxine (SDMX) Sulfonamide Antibiotic

*

* 14

b

C-Penicillin G (PENG) β-lactam Antibiotic

b

C-Thiabendazole (THIA) Fungicide Anthelmintic

a

H(G)-Ketoprofen (KETO) NSAID Analgesic

**

14

b

C-Erythromycin A (ERY) Macrolide Antibiotic

3

b

H-Ivermectin B1a (IVR) Avermectin Anthelmintic


Page 33 of 41


Figure 2. Scheme (1) of Milk partitioning processes that yielded curd and whey from skim milk (Phase 2) and retentate and permeate from whey (Phase 3).



Page 34 of 41

Figure 3. Scheme (2) of milk partitioning for determination of percent drug associated with whey or casein protein. Results indicated in boxes, OTET not assayed, as protein associated fraction too small to accurately quantify.


Page 35 of 41


Figure 4. Drug distribution and relative concentration ratios from skim milk into whey and curd fractions. Bars represent percent mean of all concentrations (n=3 concentrations; n=3 replicates per concentration) ± standard deviation of all three dose mean percentages based on disintegrations per minute (dpm) of whey and curd (at 70% moisture) fractions compared to fortified skim milk dpm. Numerical values on graph represent the mean ratio (n=3) of the drug concentration in the fraction (curd or whey) to the initial drug concentration in skim milk ± SD. Sum of stacked plots represents total, unadjusted drug recovery values.



Page 36 of 41

Figure 5. Regression analyses of observed log [Drug]curd or 0% moisture curd/[Drug]whey (log C/W or 0%mC /W) vs. log P and log D (pH 6.8) using natural y intercept. Graph A is the regression analysis of log C/W vs. log P. Graph B is the regression of log C/W vs. log D (pH 6.8). Graph C is the regression analysis of log 0%mC C/W vs. log P. Graph D is the regression of log 0%mC C/W vs. log D (pH 6.8). Drug Bank24 pKa’s accessed on 2-11-2015 (www.drugbank.ca). Log P accessed from Chemspider on 1-28-2015 (www.chemspider.com). Calculations were performed as: log Dacid = log P + log[1/(1+10pH-pKa)] or log Dbase = log P + log[1/(1+10pKa-pH)].


Page 37 of 41


Figure 6. Normalized percentages of animal drugs calculated to be in the milk end products (a) milk fat, (b) curd, (c) permeate, and (d) retentate based on data generated from the current studies. Percentage values in the curd and retentate bars represent pure curd percent and drug-to-whey protein associations normalized to whey percentages. SDMX bar has additional information on which milk end products comprise whole milk, skim milk, high-fat curd, low-fat curd, and whey, as a guide to where drug may partition during commercial milk processing.



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Figure 7. Drug distribution and relative concentration ratios of retentate and permeate fractions produced from fortified whey originating from whole milk. Bars represent percent mean of all concentrations (n=3 concentrations; n=3 replicates per concentration) ± standard deviation of the three dose means based on disintegrations per minute (dpm) of retentate and permeate fractions normalized to the total dpm between retentate and permeate. Numerical values on graph represent the mean ratios (n=3) of the drug concentration in the fraction (retentate or permeate) to the initial drug concentration in whey ± SD. Horizontal lines represent retentate and permeate percentage volumes from the filtration process. All drug distribution percentages were statistically different than volume percentages, p < 0.05.


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Figure 8. Milk partitioning scheme for all seven animal drugs, including the radiochemically assayed partitioning values, for all fractions, i.e. Phases 1 to 3. Whey and curd for Phase 2 were produced from fortified skim milk. In Phase 3, whey was produced from whole milk, then fortified and retentate and permeate fractions prepared by ultrafiltration. The wheys produced from skim milk and whole milk were similar as seen in the compositional data from Table S1. Milk fat and skim partitioning values (Phase 1) are from Hakk et al.18 The moisture content of curd was assumed to reflect drug associated with whey, and was subtracted out to obtain the “0 % Moisture Curd” value. The % of drug that was whey protein-associated was calculated by difference (total in retentate minus concentration in permeate times the concentrating factor of retentate). Phase 1

Whole Milk (100%) Fortified With OTET, PENG, ERY, SDMX, KETO, THIA, or IVR

Milk Fat (4.4% w/v) OTET: 1% PENG: 1% ERY: 2% SDMX: 5% KETO: 5% THIA: 22% IVR: 81% Skim (95% v/v) OTET: 100% PENG: 98% ERY: 97% SDMX: 92% KETO: 99% THIA: 78% IVR: 13%

0% Moisture Curd OTET: 7% PENG: 5% ERY: 16% SDMX: 22% KETO: 16% THIA: 42% IVR: 73%

Phase 2 Curd (11% w/v) OTET: 15% PENG: 12% ERY: 22% SDMX: 28% KETO: 23% THIA: 47% IVR: 75% Whey (85% v/v) OTET: 86% PENG: 85% ERY: 74% SDMX: 70% KETO: 72% THIA: 54% IVR: 21%

Phase 3 Retentate OTET: 32% PENG: 32% ERY: 39% SDMX: 39% KETO: 40% THIA: 32% IVR: 100% Permeate OTET: 68% PENG: 64% ERY: 54% SDMX: 59% KETO: 57% THIA: 62% IVR: 0.5%

ACS Paragon Plus Environment

Whey Protein Associated OTET: 8% PENG: 7% ERY: 17% SDMX: 14% KETO: 23% THIA: 12% IVR: 102%

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Distribution of Animal Drugs among Curd, Whey, and Milk Protein

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