Oxitetraciclina CA Tratament

7/25/2019 Oxitetraciclina CA Tratament

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approved treatment for these diseases in the

United States, and has been for many years.

Recently, however, strains of P. larvae

showing some tolerance to OTC have been

discovered in a limited number of beekeep-

ing operations (Shimanuki and Knox,

unpublished). This was not unexpected, as

any time there is only one treatment for adisease or other pest, there is an increased

chance of development of resistance.

1. INTRODUCTION

Oxytetracycline hydrochloride (OTC,Terramycin1) has been used since the early1950s [7, 10] for the prevention and con-trol American and European foulbrood inhoney bees (Apis mellifera L.), which are

caused by two species of bacteria, Paeni-bacillus (Bacillus) larvae andMelissococcuspluton, respectively. It is currently the only

Original article

Analysis of oxytetracycline in extender patties

Jan KOCHANSKY*

USDA ARS Bee Research Laboratory, Building 476, 10300 Baltimore Ave., Beltsville,MD 20705-2350, USA

(Received 5 August 1999; revised 15 February 2000; accepted 7 March 2000)

Abstract Antibiotic extender patties, consisting of sugar, vegetable shortening, and oxytetracycline(OTC, usually as the hydrochloride), are used for treatment/prevention of foulbrood in honey bees.An analytical method was developed to determine the concentration in the patties and to allow astudy of deterioration over time. Trituration of a sample with EDTA-treated C

18silica gel, removal

of the shortening with isooctane, and elution of the OTC with methanol/acetonitrile gave a solutionthat could be analyzed by HPLC. Different concentrations and total amounts of OTC have been rec-ommended by different authors. Not all newly-procured samples contained the same level of OTC,and the concentration decreased about 4% per year with storage at room temperature.

oxytetracycline / solid-phase extraction / HPLC / American foulbrood / European foulbrood

Apidologie 31 (2000) 517524 517 INRA/DIB-AGIB/EDP Sciences

* Correspondence and reprintsE-mail: [email protected] Mention of trade names or commercial products in this article is solely for the purpose ofproviding specific information and does not imply recommendation or endorsement by the U.S.Department of Agriculture.


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J. Kochansky518

Three methods of OTC application havebeen commonly used: a dusting with OTC inpowdered sugar repeated several times atweekly intervals [6, 8]; a solution of OTC insyrup fed to the bees [4]; and now mostcommon, an extender patty consisting ofOTC, sugar, and vegetable shortening [23,24]. The syrup formulations are undesirabledue to the instability of OTC in syrup [5,11], and the dust formulations are unpopu-lar with beekeepers since the requirementfor repeated treatments renders them toolabor-intensive for convenient use.

Early assays for OTC relied on the inhi-bition of growth of a test organism, such asErwinia sp. [6] or Bacillus cereus var.mycoides [13, 22]. While sensitive, bacteri-ological methods are incapable of distin-guishing between antibiotics, nor are theyable to assay individual compounds in mix-tures. In addition, determinations of tetra-cyclines by fluorescence measurements ofcalcium complexes have been used [1].While more selective than bacterial assays,this method was still not able to distinguishbetween OTC and other tetracyclines, most

of which fluoresce similarly.High-performance liquid chromatogra-

phy (HPLC) assays are now the preferredmethod for analysis of many substances ofbiological interest. Such methods haveallowed both good sensitivity and simulta-neous assay of various tetracyclines in honey[2, 14, 15, 17, 18, 20, 21] as well as in othersamples, such as milk and meat (for exam-ple, see [19]).

A wide variety of OTC dosages in vari-ous size extender patties have been reportedin the literature and as unpublished recom-

mendations (see below), but not always withdata supporting the efficacy of the dosagegiven. Furthermore, the amount of the pattyactually consumed by the bees, as opposedto being discarded from the hive as trash,is not known. OTC doses have ranged from200 mg [12] to 1240 mg [23, 24]. Recom-mendations during the last decade havetended toward a total of 7001000 mg of

OTC/patty, regardless of patty size [3, 16,other unpublished recommendations]. Pattysizes have ranged from 170 g (Mann-Lakeproduct label) to 590 g [3, 16] to a waxpaper covered patty was kept on the top barsthroughout the experimental period [9].OTC concentrations in patties range from1 mg/g to 5.9 mg/g (calculated from refer-ences above). This wide variation in bothconcentration and total dosage makes ananalytical method desirable for active ingre-dient in these products.

A study was conducted to determine the

OTC content of commercially-available pat-ties used for foulbrood prevention and con-trol, and to determine the stability of activeingredient in storage. In the case of extenderpatties, the large amount of grease mixedwith the antibiotic and sugar caused diffi-culties in analyses developed for normalbiological matrices. Liquid/liquid partitionfailed, since thick emulsions resulted onshaking samples with organic solvents andwater. A satisfactory separation wasachieved by a modification of the matrixsolid-phase dispersion technique of Longet al. [14, 15].

2. MATERIALS AND METHODS

2.1. Reagents and samples

New extender patty mixes were pur-chased from bee supply companies (BrushyMountain, Glorybee, Lapps Bee Supply)or had been in laboratory stock for varyinglengths of time. Short storage times wereobtained by keeping patties at room tem-perature (nominally 2025 C) for varying

lengths of time, then placing them in therefrigerator. Unless otherwise indicated, allwere stored at 5 C, and time at 5 C wasnot counted toward age. Oxytetracyclinehydrochloride and ethylenediaminete-traacetic acid (EDTA) disodium salt dihy-drate (molecular biology grade) wereobtained from Sigma. Oxalic acid dihydrate(ACS reagent grade), acetonitrile (HPLC


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run for each set of extractions and the factorobtained was used for that data set. All con-centrations in this paper are expressed asOTC hydrochloride; the concentrations ofOTC base would be 92.7% of these values.The retention time of OTC was 3.26 min(eluent ratio of 30:45:25 gave a retentiontime of 4.4 min, see below), but this becamelonger as the oxalic acid component of theeluent aged, to the point that the solutionneeded to be replaced.

2.3. Extraction and sample preparation

Initial separations of OTC from pattymixes entailed trituration of a patty sample(0.5 g) with C18 silica gel (2 g) containingEDTA disodium salt and oxalic acid (0.05 geach) in a glass mortar. This mixture wastransferred to a 10-mL plastic syringe barrelcontaining a 1.5 cm filter paper (Whatman#1). Additional C18 silica (0.5 g) was used asa dry wash of the mortar, and was then trans-ferred to the syringe barrel. After placementof another sheet of filter paper over the sil-ica, a syringe plunger (without the rubber

gasket) was used to compress the packingbed. Isooctane (10 mL) was passed throughthe column to elute the shortening. Otherhydrocarbons such as hexane or heptanewould presumably also be suitable. Afteradding ethyl acetate (2 mL, otherwise themethanol/acetonitrile eluent would not mixwith the isooctane on the column, prevent-ing flow), the OTC was eluted into a 50 mLvolumetric flask with methanol/acetonitrile(1:1) until the eluate reached the graduationmark. Aliquots of this solution were injectedonto the HPLC column.

Since the concentrations of OTC inextender patties are in the range of 16 mg/g,the concentration in the eluate solution isalso quite large. We did not investigate thedetection limits, which would be requiredfor residue assays in honey, for example.

Data reduction and plotting were per-formed with GraphPad Prism ver. 3.0(GraphPad Software, Inc. San Diego, CA).

grade), and methanol (HPLC grade) wereobtained from Aldrich. EDTA acid was atechnical grade material (Cyquest Acid)from American Cyanamid. The C18 silicagel was salvaged from unused Waters Prep500 cartridges in laboratory stock. Sincethis form of C18 silica is no longer avail-able, Supelclean LC-18 silica (Supelco) wastried and gave similar results.

Aqueous-ethanolic EDTA dipotassiumsalt was prepared from EDTA acid (29.2 g,100 mmol), KOH (13.2 g, 200 mmol assum-ing 85% assay) in water, filtration, and

refiltration after several days. Dilution to200 mL gave a 0.5M stock solution. Thisstock solution (10 mL) was diluted withwater (50 mL), ethanol (190 mL, solutioncloudy), and additional water (10 mL, solu-tion clear). The final concentration was thus19.2 mM EDTA dipotassium salt in 73%(v/v) ethanol. C

18silica gel was slurried in

this solution and the silica was filtered offwith suction and dried in air over the week-end.

2.2. HPLC conditions

Antibiotic determinations were carriedout by injection of antibiotic eluate onto a3 mm 250 mm column packed with C18silica gel (Supelcosil LC-18-DB, 5 m par-ticle size, Supelco, Inc., Bellefonte, PA),using a 20 L injector loop. Eluents weremixtures of methanol-acetonitrile-0.01 Moxalic acid. Initial ratio was 20:30:50 [19],and some later runs were carried out with aratio of 30:45:25 (see below). Flow rate was0.7 mL/min. Antibiotic concentrations weredetermined with a SpectraSYSTEMUV2000 detector attached to an SP4400

integrator (Thermo Separation Products,San Jose, CA). OTC was determined ata wavelength of 350 nm. Range was1.0 absorbance units full scale for all anal-yses. Each point is the average of three injec-tions. Injections of a series of standards inthe range of 2 ng2 g gave a linear plotwith an average detector response near 4 350detector units/ng for OTC; standards were

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J. Kochansky

3. RESULTS AND DISCUSSION

Initial experiments indicated that theshortening component of the patty could beeluted effectively with 10 mL of isooctane.The OTC was initially eluted with 10 mLof methanol/acetonitrile (1:1), but additionalsolvent removed additional analyte. Anexperiment was performed with duplicate0.5 g extender patty samples using ten suc-cessive 10-mL portions of methanol/ace-tonitrile, and analyzing each sample twice.The total OTC eluted was 2.922 0.069 mg

(mean SEM), with 2.794 0.065 mg inthe first fraction, 0.100 0.013 mg in thesecond, and 0.012 0.001, 0.006, and 0.003in fractions 35. The first 5 fractions rep-resented 99.9% of the total removed by allten fractions, so 50 mL of eluate was chosenfor all succeeding analyses.

Recovery of OTC from C18 silica gel thathad not been treated with EDTA/oxalic acidwas low and variable (only 1.151.55 mgrecovered of a 2 mg spike). Difficulties withreproducibility necessitated changes in themethod of application of the chelating agentsto the silica gel. In the initial method, EDTAand oxalic acid were added as crystallinesolids [14, 15]. This was satisfactory in thepublished cases of catfish and milk, sincethese matrices are largely aqueous and dis-tributed the chelators evenly over the silicaduring trituration. In our case, this did nothappen, and the recovery of OTC in spikedblank extender patty samples was low anduneven (1.252.08 mg recovered of 2.0 mgspike, mean recovery 1.80 0.36 SD for22 samples). Attempted dissolution ofEDTA disodium salt and oxalic acid inwater/ethanol failed, as disodium EDTA

had insufficient solubility. EDTA acid(1.96 g), oxalic acid (2.5 g) and KOH (2.16 g)were dissolved by heating in water (45 mL)and ethanol (50 mL). C18 silica gel (100 g)was added and the slurry was dried on arotary evaporator. Use of this silica mixturegave recoveries of OTC that were higherthan expected by about 10%, but the peakwas asymmetrical, with the major part

occurring about 0.15 min before the reten-tion time for OTC, with a shoulder repre-senting OTC. It seemed at the time that thetreated silica was causing decomposition ofOTC. The decomposition product was notidentified, but comparison with standardsindicated that it was not -apo-OTC or4-epi-OTC. An alternative explanation waslater found, see below.

C18 silica filter-coated with the dipotas-sium salt of EDTA gave better results. Astandard patty was prepared from sucrose(67 g), Crisco (33 g), BHT (100 mg), and

OTC hydrochloride (500 mg). Four analysesof this standard patty (4.97 mg/g OTChydrochloride) gave recoveries ranging from4.76 mg/g to 4.91 mg/g (mean SEM 4.82 0.03, mean SD 4.82 0.06) for foursamples for a mean recovery of 97.0%.

For early runs the solvent mixture usedwas methanol-acetonitrile-0.01 M oxalic acid(20:30:50 by volume) [19] at 0.7 mL/min.Later experiments used 0.7 mL/min of thesame solvents at a ratio of 30:45:25, whichgave longer retention time and baseline sep-aration from a small peak preceding the

OTC peak. With this eluent mix, however,the separation of OTC from other tetracy-clines was inferior to that given by the20:30:50 mix. The objectives of the analy-sis and the possible presence of other tetra-cyclines would determine the eluent mix-ture chosen.

One phenomenon was observed with thissystem, however; the retention time of thestandard OTC solutions was different fromthe retention times of the material elutedfrom the columns. It is likely that this isrelated to the decomposition discussed

above. Four injections of patty analyte gavea retention time of 4.220 0.014 min(mean SD) and an OTC standard gaveretention time 4.408 0.022 min. The widthat half height for both was 1.5 mm, corre-sponding to 9 seconds. An approximately1:1 mixture of the two samples, however,gave a single peak with a retention time of4.310 0.014 min, with the same half width.

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reductions to 4.05 and 3.62 min, but a sec-ond broad peak at longer retention timeappeared. Elution of 2.5 g of EDTA-treatedsilica gave 15.6 mg of residue, which fromthe observed curve would be expected toreduce the retention time by 0.2 min. Theobserved reduction was 0.19 for the run citedat the beginning of the paragraph, and0.21 min for standards run on the same dayas the EDTA curve.

OTC contents of purchased extenderpatty mixes from Manufacturer A were con-sistent with the labeled dose of 1000 mg/

patty (nominally 170 g) when purchasedfrom several distributors. A second manu-facturer did not list a concentration on thelabel, and the concentration found was lessthan in Manufacturer As product. Thesedata are shown in Table I. These data aresubject to several assumptions: when wereceived them the patties were new or hadbeen stored under conditions where nodecomposition occurred and that no decom-position occurred during our storage at 5 C.

OTC content of extender patties decreasedslowly with length of storage at room tem-

perature. Data are shown in Table I as wellas in Figure 2. The time for 25% decrease isabout 75 months (about 4%/year).

The OTC content of both commercialmixes was within the recommended rangesfor foulbrood control, and the slow decom-position at room temperature suggests thatcorrectly-mixed or commercially-prepared

If there were two compounds present, thereshould be two peaks, or at least a broaden-ing of the peak, since the separation is sig-nificantly greater than the peak width.Changing the solvent composition of thestandard had no effect on the retention time,but addition of EDTA to the standardreduced retention time proportionally toamount added. To 50 mL of OTC standard

solution containing 2.5 mg of OTChydrochloride were added amounts ofdipotassium EDTA (as a 0.5 M solution inwater) from zero to 80 mg (Fig. 1). Theretention times decreased approximately lin-early from 4.62 min with no addition to4.32 min with 20.0 mg added. Further addi-tions at levels of 40 and 80 mg gave further

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Figure 1. Change in retention time of OTC inpresence of potassium EDTA. OTC concentration

2.5 mg in 50 mL, concentrations not correctedfor changes in volume caused by EDTA addi-tions or withdrawals for injection.

Table I. OTC contents of commercial terra-patty mixes.

Sample (mg) Manufacturer mg OTC/g Suggested dose OTC/dose

1 A 6.15 0.24 patty (170 g) 1 045

2 A 5.97 0.21 patty 1 015

3 (R.T.1 ~ 9 months) A 5.79 0.15 patty 1 984

4 (R.T. ~ 2 years) A 5.72 0.06 170 g 1 972

5 (R.T. ~ 34 months) A 5.25 0.24 170 g 1 893

6 B 3.60 0.14 170226 g 6128162

1 R.T. = Room temperature (~25 C).2 The directions say to use a second patty if necessary.


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J. Kochansky

patties, stored at room temperature or below,should retain adequate antibiotic activity forseveral years. Storage at elevated tempera-tures (for example, in a closed shed out inthe sun during the summer) would beexpected to lead to more rapid inactivation.Low dosages of OTC are more likely toresult from patties improperly mixed by bee-keepers, improper storage conditions, or

from incorrect placement within the hive.Since only part of the patty is consumed,the rest being discarded by the bees, theposition in the hive is important. Wilsonet al. [23] report that Adult bees apparentlyremoved the patty, not because they enjoyedeating it, but rather because it was in theirway and annoyed them. Whenever low con-centrations of active ingredient are presentover a long period of time, whether frompatty composition (for whatever reason) orfrom placement in the hive in a locationwhere the bees take a long time to remove

the patty, the chance of resistance isincreased.

Rsum Analyse de loxyttracyclinedans les pts longue dure daction.Lhydrochlorure doxyttracycline (OTC,Terramycine) est un antibiotique utilisdepuis de nombreuses annes pour prvenir

et traiter la loque amricaine et la loqueeuropenne (NDLR : pas dAMM enFrance). Le pt longue dure daction(PLDA) est constitu de sucre, de graissevgtale et dOTC, et pos sur la tte descadres dans la ruche. Cest une mthodedapplication largement rpandue. Lesmthodes danalyse mises au point pourdtecter lOTC dans le miel ne peuvent treappliques aux PLDA, car lextraction estrendue difficile par la forte teneur en lipides.Dans ce cas la mthode de dispersion de laphase solide a t modifie afin de dtecter

lOTC. La trituration dun chantillon degel de silice C18 trait lEDTA, llimi-nation des lipides par lisooctane etllution de lOTC par le mthanol/acto-nitrile (1:1) ont permis dobtenir une solu-tion qui a pu tre analyse par chromato-graphie liquide haute pression (HPLC).Lanalyse dun mlange de PLDA prparau laboratoire a donn un taux de recouvre-ment de lOTC de 97 %. Un chantillondu commerce, libell comme contenant1 000 mg OTC/ pte, en contenait lgre-ment plus lorsquil tait frais (Tab. I), alorsquun mlange provenant dun autre four-nisseur, sans prcision de la teneur en OTC,en contenait normment moins. La teneuren OTC dcrot denviron 4 % par an en casde stockage temprature ambiante (Fig. 2).

oxyttracycline / antibiotique / loque /extraction phase solide / HPLC

Zusammenfassung Analyse von Oxyte-tracyclin in Futterteig fr Langzeitbe-handlung. Oxytetracyclin-Hydrochlorid

(OTC, Terramycin) ist seit vielen Jahren zurPrvention und zur Kontrolle von Europi-scher und Amerikanischer Faulbrut der Bie-nen in Benutzung. (redaktionelle Anmer-kung: gilt nicht fr Deutschland). Eine weitverbreitete Methode ist die Applikationdurch Langzeit Futterteig, der aus Zucker,pflanzlichem Fett und OTC besteht und deroben auf die Rhmchen gelegt wird. Die

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Figure 2. OTC extender patty storage stability at

room temperature. Extrapolation of the regressionline suggests that a 25% decrease in concentrationwould take ca. 75 months.


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[8] Gochnauer T.A., The response of normal pack-age colonies of bees to antibiotic feeding, Am.Bee J. 97 (1957) 104106.

[9] Hoopingarner R., Nelson K., American foul-brood cleanup rate using three Terramycin treat-ments, Am. Bee J. 128 (1988) 120121.

[10] Katznelson H., Arnott J., Bland S.E., Prelimi-nary report on the treatment of European foul-brood of honey bees with antibiotics, Sci. Agric.32 (1952) 180184.

[11] Kochansky J., Knox D., Shimanuki H., Com-parative stability of oxytetracycline and tylosinin sugar syrup, Apidologie 30 (1999) 321326.

[12] Lehnert T., Shimanuki H., European foulbrooddisease control in honey bee colonies used forblueberry and cranberry pollination, Am. Bee J.

120 (1980) 42930.

[13] Lehnert T., Shimanuki H., Oxytetracyclineresidues in honey following three different meth-ods of administering the antibiotic, Apidologie 12(1981) 133136.

[14] Long A.R., Hsieh L.C., Malbrough M.S., ShortC.R., Barker S.A., Matrix solid-phase disper-sion isolation and liquid chromatographic deter-mination of oxytetracycline, tetracycline, andchlortetracycline in milk, J. Assoc. Off. Anal.Chem. 73 (1990) 379384.

[15] Long A.R., Hsieh L.C., Malbrough M.S., ShortC.R., Barker S.A., Matrix solid-phase disper-sion isolation and liquid chromatographic deter-mination of oxytetracycline in catfish (Ictaluruspunctatus) muscle tissue, J. Assoc. Off. Anal.

Chem. 73 (1990) 864867.[16] Lozano L.F., Protect your bees now! Use of Ter-

ramycin in honey bee colonies, Am. Bee J. 136(1996) 572574.

[17] Matsuka M., Nakamura J., Oxytetracyclineresidues in honey and royal jelly, J. Apic. Res. 29(1990) 112117.

[18] Oka H., Ikai Y., Kawamura N., Uno K., YamadaM., Harada K.-I., Suzuki M., Improvement ofchemical analysis of antibiotics XII. Simultane-ous analysis of seven tetracyclines in honey,J. Chromatogr. 400 (1987) 253261.

[19] Oka H., Ikai Y., Hayakawa J., Masuda K.,Harada K.-I., Suzuki M., Improvement of chem-ical analysis of antibiotics. Part XIX: Determi-nation of tetracycline antibiotics in milk by liq-

uid chromatography and thin-layer chromato-graphy/fast atom bombardment mass spectrom-etry, J. Assoc. Off. Anal. Chem. Int. 77 (1994)891895.

[20] Oka H., Ikai Y., Hayakawa J., Harada K.-I.,Asukabe H., Suzuki M., Himei R., Horie M.,Nakazawa H., MacNeil J.D., Improvement ofchemical analysis of antibiotics. 22. Identificationof residual tetracyclines in honey by fritFAB/LC/MS using a volatile mobile phase,J. Agric. Food Chem. 42 (1994) 22152219.

Analyse von OTC in Honig kann nichtdirekt fr eine Analyse im Futterteig ange-wendet werden, weil die Extraktion durchden hohen Fettgehalt schwierig ist. In die-sem Fall wurde eine Modifikation derMethode der Festphasen Dispersion gewhlt,um OTC zu bestimmen. Der Futterteigprobewurde mit einem mit EDTA behandeltenC18 Silica Gel pulverisiert, danach konntedas Fett mit Isooctan entfernt werden. Diefolgende Elution von OTC aus dem Gel mitMethanol/Acetonitril (1:1) ergab eineLsung, die mit dem HPLC analysiert wer-

den konnte. Die Analyse eines frischen Fut-terteigs, der mit dem Gehalt von 1000 mgOTC/Teig gekennzeichnet war, enthieltetwas mehr OTC. Dagegen enthielt dieMischung eines anderen Anbieters, der keineGehaltsangabe gemacht hatten, wesentlichweniger. Bei einer Speicherung bei Raum-temperatur nahm der OTC Gehalt etwa 4 %pro Jahr ab.

Oxytetracyclin / Festphasenextraktion /HPLC / Faulbrut

REFERENCES

[1] Argauer R.J., Gilliam M., A fluorometric methodfor determining oxytetracycline in treated

colonies of the honey bee, Apis mell ifera

J. Invertebr. Pathol. 23 (1974) 5154.

[2] Argauer R.J., Moats W.A., Degradation of oxyte-tracycline in honey as measured by fluorescenceand liquid chromatographic assays, Apidologie22 (1991) 109115.

[3] Delaplane K.S., Lozano L.F., Using Terramycinin honey bee colonies, Am. Bee J. 134 (1994)259261.

[4] Farrar C.L., Treating bee diseases, Gleanings

Bee Cult. 84 (1956) 207211, 218.[5] Gilliam M., Argauer R.J., Stability of oxytetra-

cycline in diets fed to honeybee colonies for dis-ease control, J. Invertebr. Pathol. 26 (1975)

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[6] Girardeau J.H. Jr., The occurrence of dry-mix-fed

drugs in larval food of the honey bee, J. Econ.Entomol. 58 (1965) 878880.

[7] Gochnauer T.A., Drugs fight foulbrood diseases

in bees, Minn. Home Fm. Sci. 9 (1951) 15.

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[21] Sporns P., Kwan S., Roth L.A., HPLC analysisof oxytetracycline residues in honey, J. FoodProt. 49 (1986) 383388.

[22] Wilson W.T., Residues of oxytetracycline inhoney stored byApis mellifera, Environ. Ento-mol. 3 (1974) 674676.

[23] Wilson W.T., Elliott J.R., Lackett J.J., Antibi-otic treatments that last longer, Am. Bee J. 110(1970) 348351.

[24] Wilson W.T., Elliott J.R., Hitchcock J.D., Antibi-otic extender patties for the control of Ameri-can foul brood, J. Apic. Res. 10 (1971) 143147.

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