Simultaneous Determination of Nitroimidazoles and ...downloads.hindawi.com/journals/ijac/2018/4271385.pdf · Simultaneous Determination of Nitroimidazoles and Quinolones in Honey

Research ArticleSimultaneous Determination of Nitroimidazoles and Quinolonesin Honey by Modified QuEChERS and LC-MSMS Analysis

Haiyan Lei1 Jianbo Guo2 Zhuo Lv2 Xiaohong Zhu2 Xiaofeng Xue3

LimingWu3 andWei Cao 1

1Department of Food Science and Engineering School of Chemical Engineering Northwest University Xirsquoan Shaanxi 710069 China2Shaanxi Institute for Food and Drug Control Xirsquoan Shaanxi 710069 China3Institute of Apiculture Research Chinese Academy of Agricultural Sciences Beijing 100093 China

Correspondence should be addressed to Wei Cao caoweinwueducn

Received 2 September 2017 Revised 25 October 2017 Accepted 20 November 2017 Published 1 January 2018

Academic Editor Troy D Wood

Copyright copy 2018 Haiyan Lei et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

This study reports an analytical method for the determination of nitroimidazole and quinolones in honey using liquidchromatography-tandem mass spectrometry (LC-MSMS) A modified QuEChERS methodology was used to extract the analytesand determine veterinary drugs in honey by LC-MSMS The linear regression was excellent at the concentration levels of1ndash100 ngmL in the solution standard curve and the matrix standard curve The recovery rates of nitroimidazole and quinoloneswere 44 to 591 and 98 to 462with relative standard deviations (RSDs) below 52 and the recovery rates of nitroimidazoleand quinolones by the matrix standard curve ranged from 820 to 1178 and 79 to 1159 with relative standard deviations(RSDs) lower than 63 in acacia and jujube honey The acacia and jujube honeys have stronger matrix inhibition effect tonitroimidazole and quinolones residue the matrix inhibition effect of jujube honey is stronger than acacia honey The matrixstandard curve can calibrate matrix effect effectively In this study the detection method of antibiotics in honey can be appliedto the actual sample The results demonstrated that the modified QuEChERS method combined with LC-MSMS is a rapid highsensitive method for the analysis of nitroimidazoles and quinolones residues in honey

1 Introduction

Nitroimidazoles and quinolones (Figure 1) are a group ofantibacterial compounds that have been widely used in med-ical domainThere are many antibiotics left in honey becauseof the illegal addition of beekeepers [1ndash4] which directlythreatens the health and safety of consumers Nosemosisof bees is one of the protozoa infections in adult honey-bee which is very destructive to honeybee colonies and isan infectious disease caused by Cryptosporidium parvumNitroimidazoles for example metronidazole can be usedto prevent and treat nosemosis of bees Quinolones forinstance ofloxacin can potentially be used to prevent andtreat honeybees piroplasmosis However the misuse andillegal use of nitroimidazoles drugs may cause potentialhazards of cell mutagenicity and carcinogenic radionuclidequinolones drugs can lead to the reaction of certain degree

and hepatotoxicity or even death [5 6] Therefore nitroimi-dazoles and quinolones have been banned in honey Hencethe control of nitroimidazoles and quinolones is highly sig-nificant for the agricultural environment and food industry

High-performance liquid chromatography (HPLC) withdiode array detector (DAD) [7] liquid chromatography withfluorescence detection (LC-FD) [8] liquid chromatographic-mass spectrometric (LC-MS) [9 10] and liquid chromatog-raphy-tandem mass spectrometry (LC-MSMS) have beenused to analyze nitroimidazoles and quinolones in foodindustry (eg milk powder bovine milk butter fish tissueeggs chicken meat pig plasma bovine meat swine tissueshoney feed hair and water) [11ndash19] LC-MSMS is one ofthe most promising techniques for the analysis of antibioticsin foodstuff because of its sensitivity and accuracy Samplepreparation generally use QuEChERS approach SPE clean-up or liquid-liquid extraction Compared with SPE clean-up

HindawiInternational Journal of Analytical ChemistryVolume 2018 Article ID 4271385 12 pageshttpsdoiorg10115520184271385

2 International Journal of Analytical Chemistry

Metronidazole

N

F

O

OH

O

O

N

N

Ornidazole

N

OH

O

N

HN

O

F

N

F

O

OH

O

N

N

Ciprofloxacin

N

OH

Cl

N

O

minus

+

O

S

ONN

O

minus

+

N

N

OH

O

minus

+

Tinidazole

Ofloxacin

Enrofloxacin

Figure 1 Chemical structures of six nitroimidazoles and quinolones

and liquid-liquid extraction QuEChERS approach has al-most the same purifying effect but it requires minimumoperational steps and solvent and has higher accuracy andwider application [16 20ndash24] It was initially developed forthe analysis of pesticide residues in fruits and vegetables andwas extended to the analysis of veterinary drugs and environ-mental pollutants residues [25]

In this study we developed a multiresidue test methodbased on the application of LC-MSMS combined with mod-ified QuEChERS sample preparation methodology for rapiddetermination of nitroimidazoles and quinolones residuesThe improvement of the method is shown in Figure 2

The innovation of this method is to eliminate the matrixeffect in honey via matrix effect standard curve the matrixeffect of acacia and jujube honey has stronger inhibitoryaction to nitroimidazoles and quinolones residue with thematrix inhibition effect of jujube honey being stronger thanacacia honey The matrix standard curve can effectively cor-rect the matrix effect in honey This method is more accuratethan the previous published method

2 Experimental

21Materials and Reagents Acacia and jujube honey sampleswere purchased from consumer stores and provided by bee-keepers The samples were stored at ambient temperature(25∘C) before analysis

Analytical standard substances including metronidazole(purity = 1000) CAS 443-48-1 batch lot 100191-201507ornidazole (purity = 1000) CAS 16773-42-5 batch lot100608-201102 tinidazole (purity gt 999) CAS 19387-91-8batch lot 100336-200703 ofloxacin (purity gt 995) CAS82419-36-1 batch lot 130454-201206 ciprofloxacin (puritygt 995) CAS 85721-33-1 batch lot 130451-201203 wereobtained from Institute of Pharmaceutical and BiologicalProducts (Beijing China) enrofloxacin (purity gt 99) CAS93106-60-6 batch lot 107071 was purchased fromDr Ehren-storfer GmbH (Augsburg Germany)

These reagents of sodium chloride sodium hydroxideanhydrous sodium sulfate anhydrous magnesium sulfatecitric acid disodium hydrogen phosphate and glacial aceticacid are of analytical purity (Sinopharm Beijing China)

International Journal of Analytical Chemistry 3

10 g subsample

10 mL acetonitrile

4 g －Ａ2３4 + 1 g NaCl

Shake and centrifuge

150mg －Ａ2３4 + 25 mg PSA


(a)

3 g honey sample

5mL Mcllvaine buffer (Ｊ（ = 400)

15 mL citric acid-acetonitrile (5 95)

4 g 2３4 + 2 g NaCl


50mg ０３ + 50mg 18 + 150mg －Ａ2３4


(b)

Figure 2 Original QuEChERS methodology (a) and modified QuEChERS methodology (b)

Ammonium formate (Fluka Tianjin China) formic acid(Fluka Tianjin China) acetonitrile (Fisher Fairlawn USA)and methanol (Tedia Fairfield USA) are of HPLC gradePSA and C

18(40 120583m) are of also analytical purity (Agela

Beijing China) AMilli-Q ultrapure water system (MilliporeBedford MA USA) was used to obtain the HPLC-gradewater

Anhydrous sodium sulfate was baked at 600∘C for 3 h andmoved the sealed container for conservation

Mcilvaine buffer (pH = 400) was prepared by dissolving192 g disodium hydrogen phosphate and 89 g citric acidin 1625 L of Milli-Q water and the pH was adjusted with4molsdotLminus1 sodium hydroxide solution

22 Standard Solutions The individual stock standard solu-tions of metronidazole ornidazole tinidazole ofloxacinciprofloxacin and enrofloxacin were prepared inmethanol atthe concentration of 1mgmL The mixed working standardsolutions (1 120583gmL) were prepared by diluting stock solutionswithmethanol All standard solutions were stored atminus20∘C indark bottles

Preparation of standard solutions mixture working solu-tions (1 120583gmL) were diluted with methanolwater (5050vv) at the concentration of 1 5 10 20 40 and 100 ngmL

The matrix-matched working standard solutions 30 ghomogenized negative acacia and jujube honey samples wereweighed and placed into 50mL polypropylene centrifugetube respectively then various amounts mixture workingsolutions were added

23 Sample Preparation The modified QuEChERS method-ology was used An aliquot of 30 g homogenized samples

was weighed and placed into 50mL polypropylene centrifugetube and 5mL Mcilvaine buffer (pH = 400) was added Themixture was shaken in a vortex mixer for 30 s then 15mLcitric acid- acetonitrile (5 95) was added Subsequently 20 gsodium chloride and 40 g anhydrous sodium sulfate wereadded to this mixture and vigorously shaken in a vortex for2min afterwards the tube was centrifuged at 10000 rpm for10min Next 10mL supernatant solution was transferredinto a 15mL centrifuge tube and evaporated to 2mL undernitrogen at 40∘C Then 50mg PSA 50mg C

18 and 100mg

anhydrous Mg2SO4were added to the tube in a vortex for

2min and then centrifuged at 10000 rpm for 10min Next1mL supernatant solution was transferred into a 15mLcentrifuge tube and evaporated to dryness under nitrogen at40∘C The residue was reconstituted in 1mL methanolwater(5050 vv) and filtered through a 045 120583m filter before LC-MSMS analysis

24 LC-MSMS Conditions

241 LC Conditions Chromatographic analyses were per-formed by Waters 2695 series HPLC System (Milford MAUSA) chromatographic separation was achieved by WatersXTerra RP18 (21mmtimes 150mm 5120583m) analytical columnTheinjection volume was 10 120583L and the temperature of the col-umn was maintained at 35∘C The mobile phases were ace-tonitrile (mobile phase A) and 10mM ammonium formate +05 formic acid in Milli-Q water (mobile phase B) at a flowrate of 03mlmin The total chromatographic runtime was15min Mobile phase gradient flow program was shown inTable 1


Table 1 Mobile phase gradient flow program

Timemin A B000 5 95300 20 80800 25 75810 50 501000 50 501010 5 951500 5 95A acetonitrile B 10mM ammonium formate + 05 formic acid in Milli-Q water

Table 2 Retention time and MSMS conditions for the target compounds

Name Qualification ion (mz) Quantification ion (mz) Cone voltage (V) Collision energy (eV) ion ratio Retention time (min)

Metronidazole 17201280 17201280 25 15 369 2701720819 25

Tinidazole 2480928 2480928 30 20 663 52324801280 15

Ornidazole 22001279 22001279 30 15 645 6472200819 25

Ofloxacin 36192610 36192610 35 25 515 67236192210 35

Ciprofloxacin 33193140 33193140 40 20 317 69333192310 35

Enrofloxacin 36002450 36002450 35 25 537 73536002030 36

242 MSMS Conditions Waters Quattro Micro API triplequadruple tandem MS coupled to electrospray ionization(ESI) interface and Waters Jet Stream Ion Focusing (WatersUSA) was used for mass analysis and quantification of targetanalytes The MS was operated in the positive ion mode andutilized multiple reaction monitoring (MRM) The tuningparameters were optimized for the target analytes the gastemperature was set at 350∘C with a flow rate of 800 Lh andthe gas was high purity nitrogen Capillary voltage was 25 kVion source temperature was 120∘C cone gas flow was 50 Lhand the gas was high purity nitrogen The system operationdata acquisition and analysis are controlled and processed bythe MassHunter software

3 Results and Discussion

31 Optimization of Mass Spectrometry Conditions Bothpositive and negative ionization modes in ESI were used toevaluate the signal responses of target analytes [26] Afterevaluation the target compounds (metronidazole tinidazoleornidazole ofloxacin ciprofloxacin and enrofloxacin) wereanalysed in positive ESI mode ([M + H]+) due to higherresponse The size of collision energy and cone voltageinfluence sensitivity and fragmentation [22] In the full-scanmass analysis the parent ion is obtained then the optimumcone voltagewas optimized according to higher responseThefragmentation ion of every target compound was obtainedvia product scan in optimum cone voltage The fragment

ion of metronidazole is 1280mz and 819mz the fragmention of tinidazole is 1280mz and 928mz the fragment ionof ornidazole is 1279mz and 819mz the fragment ion ofofloxacin is 2610mz and 2210mz the fragment ion ofmetronidazole is 3140mz and 231mz and the fragmention of metronidazole is 2450mz and 203mz The collisionenergies were optimized for each individual analyte to givethe best responseThemost intense and stable fragmentationions were selected for quantification ion and the secondmost abundant ions were used for qualification ion [27]Eventually the best collision energies of quantification ionand qualification ion were optimized by the maximumintensity response All the MSMS parameters are presentedin Table 2

32 Matrix Effects Matrix is components of exclusive testedmatter which has a significant interference for linearityaccuracy precision limits of detection and quantificationThe interference was said to be matrix effect Recentlythey have been discussed in several review articles [27 28]Matrix effects (MEs) are a major problem affecting thequantitative accuracy of liquid chromatography-electrosprayionization mass spectrometry (LC-ESI-MS) when analyz-ing complicated samples [1] The influence of sample sub-strate on target compounds determination is derived fromendogenous components of sample which are organic andinorganic components and exist in extracting solution aftersample preparation the components included ion particle


25 505The concentration level of tinidazole (gkg)

0

20

40

60

80

100

120

Reco

very

()

25 505The concentration level of metronidazole (gkg)

0

20

40

60

80

100

120

Reco

very

()

25 505The concentration level of ofloxacin (gkg)

0

20

40

60

80

100

120

Reco

very

()

25 505The concentration level of ornidazole (gkg)

0

20

40

60

80

100

120

Reco

very

()

25 505The concentration level of ciprofloxacin (gkg)

20

40

60

80

100

120

Reco

very

()

25 505The concentration level of enrofloxacin (gkg)

0

20

40

60

80

100

120

Reco

very

()

Figure 3 Absolute and relative recovery of target compounds ◼e the recovery rates of six kinds of drugs by the solution standard curve inacacia and jujube honey respectively 998787 998771 the recovery rates of six kinds of drugs by the matrix standard curve in acacia and jujube honeyrespectively

composition (electrolytes and salts) strong polar compounds(phenols and pigment) and organic components (sugarsamines carbamide lipoid congener and metabolism oftarget object) when these substances and target compoundsfly into the ion source at the same time after honey samplespreparation they will affect the ionization process of targetcompounds [29] In the study the matrix effects in thesesamples were evaluated by the spiked honey samples at 525 and 50 120583gkg According to Figure 3 compared with

matrix-matched calibration curve the recovery of the neatstandard calibration curve was lower and ME indicatedmatrix suppression ME phenomena were obvious in honeysample whichmay be impressed by sugars phenols pigmentprotein and flavonoids Moreover jujube honey containhigh contents of pigment and flavonoids that make it darktherefore recovery of jujube honey is lower than acaciahoney and the matrix effect of jujube honey was strongerthan acacia honey Hence matrix-matched standard curve


QAA1269 24-Nov-2016

Time100 200 300 400 500 600 700 800

1110-YHHS-1 1 MRM of 2 Channels ES +TIC

203e427073

0

100

()


143e4693186

24165

0124 379

1020

100

()

200 300 400 500 600 700 800100Time


260e4735197

0

100

()

200 300 400 500 600 700 800100Time


574e4672180

0

100

()

200 300 400 500 600 700 800100Time


132e4523140

0154

377101

23964

392105

0

100

()

200 300 400 500 600 700 800100Time


218e4647173

0

100(

)

200 300 400 500 600 700 800100Time

204638 24-Nov-2016

Figure 4 Typical chromatograms of MRM transitions for metronidazole tinidazole ornidazole ofloxacin ciprofloxacin and enrofloxacinat the concentration of 25 120583gkg in acacia honey

has been used to overcome the matrix effect and signalirreproducibility matrix interference and loss of recoveryFigure 3 showed that the matrix-matched working standardcurves for each compound can meet detection requirementsand effectively correct matrix effects in acacia and jujubehoney

33 Linearity A serial dilution of the standard mixture wasprepared (1ndash100 ngmL) and analyzed using the optimizedassay conditions And the correlation coefficients (1198772) rangedwithin 0994ndash0999 using this method The negative acaciaand jujube honeys were extracted and tested according toSections 23 and 24 The matrix-matched calibration curve

was constructed by determining the peak area of metron-idazole ornidazole tinidazole ofloxacin ciprofloxacin andenrofloxacin at six concentration levels in the range of10ndash100 ngmL curve in acacia and jujube honey and thecorrelation coefficients (1198772) ranged within 0990ndash0999 and0992ndash0996 respectively In general the linearity of thematrix-matched working standard curves and the solutionstandard curve was excellent The data were treated by usingtheWaters QuanLynx module of the MassLynx softwareTheresult is shown in Table 3

34 Limit of Detection and Quantification The limits ofdetection (LOD) and limits of quantification (LOQ) were


Table3Linear

rangeregressio

nequatio

nandcorrela

tioncoeffi

cients

Com

poun

dLinear

rang

e(ng

mL)

Calib

ratio

nequatio

nCorrelationcoeffi

cients1198772

Standard

solutio

nsAc

aciaho

ney

Jujube

honey

Standard

solutio

nsAc

aciaho

ney

Jujube

honey

Metronidazole

10ndash100

119910=149667119909+228847119910=842987119909+173078119910=366167119909+330998

0998

0997

0996

Tinidazole

10ndash100

119910=693581119909+468099119910=471843119909minus590018119910=335304119909+83281

0997

0997

0993

Ornidazole

10ndash100

119910=867363119909+1629119910=578037119909+89570119910=381225119909+135625

0999

0999

0992

Oflo

xacin

10ndash100

119910=159216119909minus487671119910=622512119909+566425119910=512493119909+52237

0994

0990

0993

Ciprofl

oxacin

10ndash100

119910=799202119909minus207609119910=408103119909+42569119910=330771119909+255369

0996

0999

0996

Enroflo

xacin

10ndash100

119910=884074119909minus347422119910=691707119909+748835119910=429161119909+120416

0995

0998

0992


QAA1269 24-Nov-2016

1110-ZHHS-1TIC

849e327073

16946

386104

200 300 400 500 600 700 800100Time

0

100

()

1110-ZHHS-1TIC

545e3697187

22260 659

177454122

386104

644173

615165

723194

764205

200 300 400 500 600 700 800100Time

0

100

()

1110-ZHHS-1TIC

161e4739198

765205

200 300 400 500 600 700 800100Time

0

100

()

1110-ZHHS-1TIC

254e4672180

200 300 400 500 600 700 800100Time

0

100

()

1110-ZHHS-1TIC

889e3527141

389104

13436

200 300 400 500 600 700 800100Time

0

100

()

1110-ZHHS-1TIC

129e4647173

200 300 400 500 600 700 800100Time

0

100

()

224347 24-Nov-20166 MRM of 2 Channels ES +

5 MRM of 3 Channels ES +





Figure 5 Typical chromatograms of MRM transitions for metronidazole tinidazole ornidazole ofloxacin ciprofloxacin and enrofloxacinat the concentration of 25 120583gkg in jujube honey

determined (119899 = 3) via the signal-to-noise ratio (SNR)of the analyte LOD and LOQ were estimated as 3 timesSN and 10 times SN respectively The LODs of the targetcompounds were achieved in the range of 064ndash141120583gkgand 083ndash158 120583gkg for acacia and jujube honey respec-tively The LOQs of the target compounds were achievedin the range of 213ndash470 120583gkg and 277ndash527 120583gkg foracacia and jujube honey respectively The result is shown inTable 4

35 Recovery and Precision Recovery and precision weredetermined at three concentration levels (5 25 and 50120583gkg)

for acacia and jujube honey Recoveries of the analytesranged within 819ndash1168 and 810ndash1159 in acaciaand jujube honey respectively In detail recoveries rangewithin 810minus1007 (metronidazole) 825ndash1156 (tin-idazole) 852ndash1133 (ornidazole) 819ndash1167 (oflox-acin) 818ndash1168 (ciprofloxacin) and 865ndash1159(enrofloxacin) Recoveries for target compounds were higherthan 80 and RSDs were lower than 63 in Table 5 Theseare highly acceptable values for the honey samples Typicalchromatograms of MRM transitions about six drugs areshown in Figures 4 and 5 at the concentration of 25 120583gkg inacacia and jujube honey


Table 4 LODs and LOQs for the target compounds

Compound Acacia honey Jujube honeyLOD(120583gkg) LOQ(120583gkg) LOD(120583gkg) LOQ(120583gkg)

Metronidazole 064 213 083 277Tinidazole 125 417 151 503Ornidazole 141 470 158 527Ofloxacin 077 256 099 330Ciprofloxacin 092 307 117 391Enrofloxacin 113 376 124 413

Table 5 Recovery rates and RSDs of target compounds from honey samples using by LC-MSMS

Compound Concentration level (120583gkg) Recovery (RSD)Acacia honey Jujube honey

Metronidazole5 827 (0044) 810 (0058)25 857 (0031) 1007 (0050)50 861 (0020) 990 (0024)

Tinidazole5 1156 (0050) 8560 (0027)25 872 (0031) 1005 (0051)50 826 (0012) 825 (0039)

Ornidazole5 1133 (0044) 868 (0017)25 932 (0049) 1018 (0036)50 852 (0043) 1089 (0007)

Ofloxacin5 1167 (0046) 829 (0057)25 886 (0054) 948 (0039)50 819 (0019) 1065 (0023)

Ciprofloxacin5 831 (0027) 831 (0027)25 968 (0040) 968 (0040)50 1168 (0050) 818 (0033)

Enrofloxacin5 1099 (0053) 865 (0063)25 1159 (0044) 1039 (0062)50 1120 (0031) 1159 (0020)

The repeatability and intermediate precision results were expressed as relative standard deviation (RSD) number of determinations (119899) = 3

36 Application to Real Samples The proposed modifiedQuEChERS method with LC-MSMS was applied to 46actual honey samples fromhoney producers and cooperativeslocated in the cities of Shaanxi Hebei Gansu ChongqingHubei and Shanxi in China The samples were extracted andanalysed according to the protocols described in Sections23 and 24 Quantifications of metronidazole tinidazoleornidazole ofloxacin ciprofloxacin and enrofloxacin wereperformed using the external standardThe results are shownin Table 6

4 Conclusion

A simple and rapid method of modified QuEChERS com-bined with liquid chromatography-tandem mass spectrom-etry for the determination of multiresidue in honey sampleswas established The method was fast efficient reliable andcould be used in the monitoring of antibiotic in honeywhich is fit for the purpose and satisfactory in terms of

selectivity recovery and accuracy Therefore it has beensuccessfully applied to the determination of nitroimidazolesand quinolones residues Matrix standard calibration curveswere successfully employed in correction for matrix effectIt can be seen that some honeys have the residues ofnitroimidazole and quinolones in Table 6 Hence the controlof antibiotics in food is highly significant for the agriculturalenvironment and food industry

Conflicts of Interest

The authors declare that they have no conflicts of interest

Acknowledgments

Thiswork is financially supported by the Agricultural Scienceand Technology Innovation Program of Shaanxi Province(2012NKC01-21) the Agricultural Science and TechnologyProject of Shaanxi Province (2013K01-47-01) and Social


Table 6 Concentrations of antibiotics detected in acacia and jujube honey

Number Concentration of target compounds (120583gkg) (119899 = 3)Metronidazole Tinidazole Ornidazole Ofloxacin Ciprofloxacin Enrofloxacin

(1) ND ND ND ND ND ND(2) ND ND ND ND ND ND(3) 6695 ND ND ND ND ND(4) ND ND ND ND ND ND(5) ND ND ND ND ND ND(6) ND ND ND ND ND ND(7) ND ND ND ND ND ND(8) ND ND ND ND ND ND(9) ND ND ND ND ND ND(10) ND ND ND ND ND ND(11) ND ND ND ND 8943 ND(12) ND ND ND ND ND ND(13) ND ND ND ND ND ND(14) ND ND ND ND ND ND(15) ND ND ND ND ND ND(16) ND ND ND ND ND ND(17) ND ND ND ND ND ND(18) ND ND ND ND ND ND(19) ND ND ND ND ND ND(20) ND ND ND ND ND ND(21) ND ND ND ND ND ND(22) ND ND ND ND ND ND(23) ND ND ND ND ND ND(24) ND ND ND ND ND ND(25) ND ND ND ND ND ND(26) ND ND ND ND ND ND(27) ND ND ND ND ND ND(28) 587 ND ND ND ND ND(29) ND ND ND ND ND ND(30) ND ND ND ND ND ND(31) ND ND ND ND ND ND(32) ND ND ND ND ND ND(33) ND ND ND ND ND ND(34) ND ND ND ND ND ND(35) 2935 ND ND ND 5291 ND(36) ND ND ND ND ND ND(37) ND ND ND ND ND ND(38) ND ND ND ND ND ND(39) ND ND ND ND ND ND(40) ND ND ND ND ND ND(41) ND ND ND ND ND ND(42) ND ND ND ND ND ND(43) 612 ND ND ND ND ND(44) ND ND ND ND ND ND(45) ND ND ND ND ND NDND not detected numbers (1)ndash(33) acacia honey numbers (34)ndash(45) jujube honey


Development of Science and Technology Project (2016SF-425)

References

[1] A H Shendy M A Al-Ghobashy S A Gad Alla and H MLotfy ldquoDevelopment and validation of a modified QuEChERSprotocol coupled to LC-MSMS for simultaneous determina-tion ofmulti-class antibiotic residues in honeyrdquo FoodChemistryvol 190 pp 982ndash989 2016

[2] J M Płotka M Biziuk and C Morrison ldquoDetermination ofantibiotic residues in honeyrdquo Trends in Analytical Chemistryvol 30 pp 1035ndash1041 2011

[3] P Butaye L A Devriese and F Haesebrouck ldquoDifferencesin antibiotic resistance patterns of Enterococcus faecalis andEnterococcus faecium strains isolated from farm and petanimalsrdquo Antimicrobial Agents and Chemotherapy vol 45 no5 pp 1374ndash1378 2001

[4] R Venable C Haynes and J M Cook ldquoReported prevalenceand quantitative LC-MS methods for the analysis of veterinarydrug residues in honey A reviewrdquo Food Additives amp Contam-inants Part A Chemistry Analysis Control Exposure amp RiskAssessment vol 31 no 4 pp 621ndash640 2014

[5] Y Z Chen and B Hu ldquoThe harm of veterinary drug residuesin animal foods and its reason analysisrdquo Journal of Food andBiological Technology vol 28 pp 162ndash166 2009

[6] M Y Chen T Sun and X M Wang ldquoVeterinary drug residueand its risksrdquo Progress in Animal Medicine vol 26 pp 111ndash1132005

[7] Y-J Yu H-L Wu S-Z Shao et al ldquoUsing second-order cal-ibration method based on trilinear decomposition algorithmscoupled with high performance liquid chromatography withdiode array detector for determination of quinolones in honeysamplesrdquo Talanta vol 85 no 3 pp 1549ndash1559 2011

[8] D Terrado-Campos K Tayeb-Cherif J Peris-Vicente S Carda-Broch and J Esteve-Romero ldquoDetermination of oxolinic aciddanofloxacin ciprofloxacin and enrofloxacin in porcine andbovine meat by micellar liquid chromatography with fluores-cence detectionrdquo Food Chemistry vol 221 pp 1277ndash1284 2017

[9] A Di Corcia and M Nazzari ldquoLiquid chromatographic-massspectrometric methods for analyzing antibiotic and antibacte-rial agents in animal food productsrdquo Journal of ChromatographyA vol 974 no 1-2 pp 53ndash89 2002

[10] W-X Zhu J-Z Yang Z-X Wang C-J Wang Y-F Liu andL Zhang ldquoRapid determination of 88 veterinary drug residuesin milk using automated TurborFlow online clean-up modecoupled to liquid chromatography-tandemmass spectrometryrdquoTalanta vol 148 pp 401ndash411 2016

[11] M E Dasenaki and N S Thomaidis ldquoMulti-residue deter-mination of 115 veterinary drugs and pharmaceutical residuesin milk powder butter fish tissue and eggs using liquidchromatography-tandem mass spectrometryrdquo Analytica Chim-ica Acta vol 880 pp 103ndash121 2015

[12] X Xia Y Wang X Wang et al ldquoValidation of a methodfor simultaneous determination of nitroimidazoles benzimida-zoles and chloramphenicols in swine tissues by ultra-high per-formance liquid chromatography-tandem mass spectrometryrdquoJournal of Chromatography A vol 1292 pp 96ndash103 2013

[13] R Galarini G Saluti D Giusepponi R Rossi and S MorettildquoMulticlass determination of 27 antibiotics in honeyrdquo FoodControl vol 48 pp 12ndash24 2015

[14] A Tolgyesi V K Sharma S Fekete J Fekete A Simon and SFarkas ldquoDevelopment of a rapid method for the determinationand confirmation of nitroimidazoles in six matrices by fastliquid chromatography-tandem mass spectrometryrdquo Journal ofPharmaceutical and Biomedical Analysis vol 64-65 pp 40ndash482012

[15] A Gadaj V Di Lullo H Cantwell M McCormack A Fureyand M Danaher ldquoDetermination of nitroimidazole residues inaquaculture tissue using ultra high performance liquid chro-matography coupled to tandem mass spectrometryrdquo Journal ofChromatography B vol 960 pp 105ndash115 2014

[16] G Stubbings and T Bigwood ldquoThe development and vali-dation of a multiclass liquid chromatography tandem massspectrometry (LC-MSMS) procedure for the determinationof veterinary drug residues in animal tissue using a quechers(Quick Easy Cheap Effective Rugged and Safe) approachrdquoAnalytica Chimica Acta vol 637 no 1-2 pp 68ndash78 2009

[17] K Bousovaa H Senyuva and K Mittendorf ldquoQuantitativemulti-residue method for determination antibiotics in chickenmeat using turbulent flow chromatography coupled to liquidchromatography-tandem mass spectrometryrdquo Journal of Chro-matography A vol 1274 pp 19ndash27 2013

[18] L R Guidi F A Santos A C S R Ribeiro C FernandesL H M Silva and M B A Gloria ldquoA simple fast andsensitive screening LC-ESI-MSMS method for antibiotics infishrdquo Talanta vol 163 pp 85ndash93 2017

[19] M T Martins F Barreto R B Hoff et al ldquoMulticlass andmulti-residue determination of antibiotics in bovine milk byliquid chromatography-tandem mass spectrometry Combin-ing efficiency of milk control and simplicity of routine analysisrdquoInternational Dairy Journal vol 59 pp 44ndash51 2016

[20] B K Matuszewski M L Constanzer and C M Chavez-EngldquoStrategies for the assessment of matrix effect in quantitativebioanalytical methods based on HPLC-MSMSrdquo AnalyticalChemistry vol 75 no 13 pp 3019ndash3030 2003

[21] A Posyniak J Zmudzki and K Mitrowska ldquoDispersive solid-phase extraction for the determination of sulfonamides inchicken muscle by liquid chromatographyrdquo Journal of Chro-matography A vol 1087 no 1-2 pp 259ndash264 2005

[22] G Chen P Cao and R Liu ldquoA multi-residue method forfast determination of pesticides in tea by ultra performanceliquid chromatography-electrospray tandem mass spectrome-try combined with modified QuEChERS sample preparationprocedurerdquo Food Chemistry vol 125 no 4 pp 1406ndash1411 2011

[23] M Anastassiades S J Lehotay D Stajnbaher and F J SchenckldquoFast and easy multiresidue method employing acetonitrileextractionpartitioning and ldquodispersive solid-phase extractionrdquofor the determination of pesticide residues in producerdquo Journalof AOAC International vol 86 no 2 pp 412ndash431 2003

[24] K H Park J-H Choi A M Abd El-Aty et al ldquoQuanti-fying fenobucarb residue levels in beef muscles using liquidchromatography-tandem mass spectrometry and QuEChERSsample preparationrdquo Food Chemistry vol 138 no 4 pp 2306ndash2311 2013

[25] M Tenon N Feuillere M Roller and S Birtic ldquoDevelopmentand validation of a multiclass method for the quantificationof veterinary drug residues in honey and royal jelly by liquidchromatography-tandem mass spectrometryrdquo Food Chemistryvol 221 pp 1298ndash1307 2017

[26] H C Liu T Lin andX L Cheng ldquoSimultaneous determinationof anabolic steroids and 120573-agonists in milk by QuEChERS andultra high performance liquid chromatography tandem mass


spectrometryrdquo Journal of Chromatography B vol 1043 pp 176ndash186 2017

[27] V Gresslera A R L Franzenb G J M M de Lima F CTavernari O A D Costa and V Feddern ldquoDevelopment of areadily appliedmethod to quantify ractopamine residue inmeatand bonemeal by quechers-lc-msmsrdquo Journal of Chromatogra-phy B vol 1015-1016 pp 192ndash200 2016

[28] H-W Liao G-Y Chen I-L Tsai and C-H Kuo ldquoUsing apostcolumn-infused internal standard for correcting thematrixeffects of urine specimens in liquid chromatography-electro-spray ionization mass spectrometryrdquo Journal of Chromatogra-phy A vol 1327 pp 97ndash104 2014

[29] L Q Wang L M He Z L Zeng and J X Chen ldquoStudy ofmatrix effects for liquid chromatography tandem mass spec-trometry analysis of veterinary drug residuesrdquo Journal of MassSpectrometry vol 32 pp 321ndash331 2011

TribologyAdvances in

Hindawiwwwhindawicom Volume 2018


International Journal ofInternational Journal ofPhotoenergy


Journal of

Chemistry


Advances inPhysical Chemistry

Hindawiwwwhindawicom

Analytical Methods in Chemistry

Journal of

Volume 2018

Bioinorganic Chemistry and ApplicationsHindawiwwwhindawicom Volume 2018

SpectroscopyInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

Volume 2018

Medicinal ChemistryInternational Journal of


NanotechnologyHindawiwwwhindawicom Volume 2018

Journal of

Applied ChemistryJournal of



Biochemistry Research International


Enzyme Research


Journal of

SpectroscopyAnalytical ChemistryInternational Journal of


MaterialsJournal of



BioMed Research International Electrochemistry

International Journal of


Na

nom

ate

ria

ls


Journal ofNanomaterials

Submit your manuscripts atwwwhindawicom


Metronidazole

N

F

O

OH

O

O

N

N

Ornidazole

N

OH

O

N

HN

O

F

N

F

O

OH

O

N

N

Ciprofloxacin

N

OH

Cl

N

O

minus

+

O

S

ONN

O

minus

+

N

N

OH

O

minus

+

Tinidazole

Ofloxacin

Enrofloxacin

Figure 1 Chemical structures of six nitroimidazoles and quinolones

and liquid-liquid extraction QuEChERS approach has al-most the same purifying effect but it requires minimumoperational steps and solvent and has higher accuracy andwider application [16 20ndash24] It was initially developed forthe analysis of pesticide residues in fruits and vegetables andwas extended to the analysis of veterinary drugs and environ-mental pollutants residues [25]

In this study we developed a multiresidue test methodbased on the application of LC-MSMS combined with mod-ified QuEChERS sample preparation methodology for rapiddetermination of nitroimidazoles and quinolones residuesThe improvement of the method is shown in Figure 2

The innovation of this method is to eliminate the matrixeffect in honey via matrix effect standard curve the matrixeffect of acacia and jujube honey has stronger inhibitoryaction to nitroimidazoles and quinolones residue with thematrix inhibition effect of jujube honey being stronger thanacacia honey The matrix standard curve can effectively cor-rect the matrix effect in honey This method is more accuratethan the previous published method

2 Experimental

21Materials and Reagents Acacia and jujube honey sampleswere purchased from consumer stores and provided by bee-keepers The samples were stored at ambient temperature(25∘C) before analysis

Analytical standard substances including metronidazole(purity = 1000) CAS 443-48-1 batch lot 100191-201507ornidazole (purity = 1000) CAS 16773-42-5 batch lot100608-201102 tinidazole (purity gt 999) CAS 19387-91-8batch lot 100336-200703 ofloxacin (purity gt 995) CAS82419-36-1 batch lot 130454-201206 ciprofloxacin (puritygt 995) CAS 85721-33-1 batch lot 130451-201203 wereobtained from Institute of Pharmaceutical and BiologicalProducts (Beijing China) enrofloxacin (purity gt 99) CAS93106-60-6 batch lot 107071 was purchased fromDr Ehren-storfer GmbH (Augsburg Germany)

These reagents of sodium chloride sodium hydroxideanhydrous sodium sulfate anhydrous magnesium sulfatecitric acid disodium hydrogen phosphate and glacial aceticacid are of analytical purity (Sinopharm Beijing China)


10 g subsample

10 mL acetonitrile

4 g －Ａ2３4 + 1 g NaCl


150mg －Ａ2３4 + 25 mg PSA


(a)

3 g honey sample



4 g 2３4 + 2 g NaCl


50mg ０３ + 50mg 18 + 150mg －Ａ2３4


(b)












18 and 100mg










Metronidazole 17201280 17201280 25 15 369 2701720819 25

Tinidazole 2480928 2480928 30 20 663 52324801280 15

Ornidazole 22001279 22001279 30 15 645 6472200819 25

Ofloxacin 36192610 36192610 35 25 515 67236192210 35

Ciprofloxacin 33193140 33193140 40 20 317 69333192310 35

Enrofloxacin 36002450 36002450 35 25 537 73536002030 36








0

20

40

60

80

100

120

Reco

very

()


0

20

40

60

80

100

120

Reco

very

()


0

20

40

60

80

100

120

Reco

very

()


0

20

40

60

80

100

120

Reco

very

()


20

40

60

80

100

120

Reco

very

()


0

20

40

60

80

100

120

Reco

very

()





QAA1269 24-Nov-2016

Time100 200 300 400 500 600 700 800


203e427073

0

100

()


143e4693186

24165

0124 379

1020

100

()

200 300 400 500 600 700 800100Time


260e4735197

0

100

()

200 300 400 500 600 700 800100Time


574e4672180

0

100

()

200 300 400 500 600 700 800100Time


132e4523140

0154

377101

23964

392105

0

100

()

200 300 400 500 600 700 800100Time


218e4647173

0

100(

)

200 300 400 500 600 700 800100Time

204638 24-Nov-2016







Table3Linear

rangeregressio

nequatio

nandcorrela

tioncoeffi

cients

Com

poun

dLinear

rang

e(ng

mL)

Calib

ratio

nequatio

nCorrelationcoeffi

cients1198772

Standard

solutio

nsAc

aciaho

ney

Jujube

honey

Standard

solutio

nsAc

aciaho

ney

Jujube

honey

Metronidazole

10ndash100

119910=149667119909+228847119910=842987119909+173078119910=366167119909+330998

0998

0997

0996

Tinidazole

10ndash100

119910=693581119909+468099119910=471843119909minus590018119910=335304119909+83281

0997

0997

0993

Ornidazole

10ndash100

119910=867363119909+1629119910=578037119909+89570119910=381225119909+135625

0999

0999

0992

Oflo

xacin

10ndash100

119910=159216119909minus487671119910=622512119909+566425119910=512493119909+52237

0994

0990

0993

Ciprofl

oxacin

10ndash100

119910=799202119909minus207609119910=408103119909+42569119910=330771119909+255369

0996

0999

0996

Enroflo

xacin

10ndash100

119910=884074119909minus347422119910=691707119909+748835119910=429161119909+120416

0995

0998

0992


QAA1269 24-Nov-2016

1110-ZHHS-1TIC

849e327073

16946

386104

200 300 400 500 600 700 800100Time

0

100

()

1110-ZHHS-1TIC

545e3697187

22260 659

177454122

386104

644173

615165

723194

764205

200 300 400 500 600 700 800100Time

0

100

()

1110-ZHHS-1TIC

161e4739198

765205

200 300 400 500 600 700 800100Time

0

100

()

1110-ZHHS-1TIC

254e4672180

200 300 400 500 600 700 800100Time

0

100

()

1110-ZHHS-1TIC

889e3527141

389104

13436

200 300 400 500 600 700 800100Time

0

100

()

1110-ZHHS-1TIC

129e4647173

200 300 400 500 600 700 800100Time

0

100

()

















Metronidazole5 827 (0044) 810 (0058)25 857 (0031) 1007 (0050)50 861 (0020) 990 (0024)

Tinidazole5 1156 (0050) 8560 (0027)25 872 (0031) 1005 (0051)50 826 (0012) 825 (0039)

Ornidazole5 1133 (0044) 868 (0017)25 932 (0049) 1018 (0036)50 852 (0043) 1089 (0007)

Ofloxacin5 1167 (0046) 829 (0057)25 886 (0054) 948 (0039)50 819 (0019) 1065 (0023)

Ciprofloxacin5 831 (0027) 831 (0027)25 968 (0040) 968 (0040)50 1168 (0050) 818 (0033)

Enrofloxacin5 1099 (0053) 865 (0063)25 1159 (0044) 1039 (0062)50 1120 (0031) 1159 (0020)



4 Conclusion





Acknowledgments








References





































Journal of

Chemistry





Journal of

Volume 2018






Volume 2018




Journal of






Enzyme Research


Journal of



MaterialsJournal of






Na

nom

ate

ria

ls





10 g subsample

10 mL acetonitrile

4 g －Ａ2３4 + 1 g NaCl


150mg －Ａ2３4 + 25 mg PSA


(a)

3 g honey sample



4 g 2３4 + 2 g NaCl


50mg ０３ + 50mg 18 + 150mg －Ａ2３4


(b)












18 and 100mg










Metronidazole 17201280 17201280 25 15 369 2701720819 25

Tinidazole 2480928 2480928 30 20 663 52324801280 15

Ornidazole 22001279 22001279 30 15 645 6472200819 25

Ofloxacin 36192610 36192610 35 25 515 67236192210 35

Ciprofloxacin 33193140 33193140 40 20 317 69333192310 35

Enrofloxacin 36002450 36002450 35 25 537 73536002030 36








0

20

40

60

80

100

120

Reco

very

()


0

20

40

60

80

100

120

Reco

very

()


0

20

40

60

80

100

120

Reco

very

()


0

20

40

60

80

100

120

Reco

very

()


20

40

60

80

100

120

Reco

very

()


0

20

40

60

80

100

120

Reco

very

()





QAA1269 24-Nov-2016

Time100 200 300 400 500 600 700 800


203e427073

0

100

()


143e4693186

24165

0124 379

1020

100

()

200 300 400 500 600 700 800100Time


260e4735197

0

100

()

200 300 400 500 600 700 800100Time


574e4672180

0

100

()

200 300 400 500 600 700 800100Time


132e4523140

0154

377101

23964

392105

0

100

()

200 300 400 500 600 700 800100Time


218e4647173

0

100(

)

200 300 400 500 600 700 800100Time

204638 24-Nov-2016







Table3Linear

rangeregressio

nequatio

nandcorrela

tioncoeffi

cients

Com

poun

dLinear

rang

e(ng

mL)

Calib

ratio

nequatio

nCorrelationcoeffi

cients1198772

Standard

solutio

nsAc

aciaho

ney

Jujube

honey

Standard

solutio

nsAc

aciaho

ney

Jujube

honey

Metronidazole

10ndash100

119910=149667119909+228847119910=842987119909+173078119910=366167119909+330998

0998

0997

0996

Tinidazole

10ndash100

119910=693581119909+468099119910=471843119909minus590018119910=335304119909+83281

0997

0997

0993

Ornidazole

10ndash100

119910=867363119909+1629119910=578037119909+89570119910=381225119909+135625

0999

0999

0992

Oflo

xacin

10ndash100

119910=159216119909minus487671119910=622512119909+566425119910=512493119909+52237

0994

0990

0993

Ciprofl

oxacin

10ndash100

119910=799202119909minus207609119910=408103119909+42569119910=330771119909+255369

0996

0999

0996

Enroflo

xacin

10ndash100

119910=884074119909minus347422119910=691707119909+748835119910=429161119909+120416

0995

0998

0992


QAA1269 24-Nov-2016

1110-ZHHS-1TIC

849e327073

16946

386104

200 300 400 500 600 700 800100Time

0

100

()

1110-ZHHS-1TIC

545e3697187

22260 659

177454122

386104

644173

615165

723194

764205

200 300 400 500 600 700 800100Time

0

100

()

1110-ZHHS-1TIC

161e4739198

765205

200 300 400 500 600 700 800100Time

0

100

()

1110-ZHHS-1TIC

254e4672180

200 300 400 500 600 700 800100Time

0

100

()

1110-ZHHS-1TIC

889e3527141

389104

13436

200 300 400 500 600 700 800100Time

0

100

()

1110-ZHHS-1TIC

129e4647173

200 300 400 500 600 700 800100Time

0

100

()

















Metronidazole5 827 (0044) 810 (0058)25 857 (0031) 1007 (0050)50 861 (0020) 990 (0024)

Tinidazole5 1156 (0050) 8560 (0027)25 872 (0031) 1005 (0051)50 826 (0012) 825 (0039)

Ornidazole5 1133 (0044) 868 (0017)25 932 (0049) 1018 (0036)50 852 (0043) 1089 (0007)

Ofloxacin5 1167 (0046) 829 (0057)25 886 (0054) 948 (0039)50 819 (0019) 1065 (0023)

Ciprofloxacin5 831 (0027) 831 (0027)25 968 (0040) 968 (0040)50 1168 (0050) 818 (0033)

Enrofloxacin5 1099 (0053) 865 (0063)25 1159 (0044) 1039 (0062)50 1120 (0031) 1159 (0020)



4 Conclusion





Acknowledgments








References





































Journal of

Chemistry





Journal of

Volume 2018






Volume 2018




Journal of






Enzyme Research


Journal of



MaterialsJournal of






Na

nom

ate

ria

ls









Metronidazole 17201280 17201280 25 15 369 2701720819 25

Tinidazole 2480928 2480928 30 20 663 52324801280 15

Ornidazole 22001279 22001279 30 15 645 6472200819 25

Ofloxacin 36192610 36192610 35 25 515 67236192210 35

Ciprofloxacin 33193140 33193140 40 20 317 69333192310 35

Enrofloxacin 36002450 36002450 35 25 537 73536002030 36








0

20

40

60

80

100

120

Reco

very

()


0

20

40

60

80

100

120

Reco

very

()


0

20

40

60

80

100

120

Reco

very

()


0

20

40

60

80

100

120

Reco

very

()


20

40

60

80

100

120

Reco

very

()


0

20

40

60

80

100

120

Reco

very

()





QAA1269 24-Nov-2016

Time100 200 300 400 500 600 700 800


203e427073

0

100

()


143e4693186

24165

0124 379

1020

100

()

200 300 400 500 600 700 800100Time


260e4735197

0

100

()

200 300 400 500 600 700 800100Time


574e4672180

0

100

()

200 300 400 500 600 700 800100Time


132e4523140

0154

377101

23964

392105

0

100

()

200 300 400 500 600 700 800100Time


218e4647173

0

100(

)

200 300 400 500 600 700 800100Time

204638 24-Nov-2016







Table3Linear

rangeregressio

nequatio

nandcorrela

tioncoeffi

cients

Com

poun

dLinear

rang

e(ng

mL)

Calib

ratio

nequatio

nCorrelationcoeffi

cients1198772

Standard

solutio

nsAc

aciaho

ney

Jujube

honey

Standard

solutio

nsAc

aciaho

ney

Jujube

honey

Metronidazole

10ndash100

119910=149667119909+228847119910=842987119909+173078119910=366167119909+330998

0998

0997

0996

Tinidazole

10ndash100

119910=693581119909+468099119910=471843119909minus590018119910=335304119909+83281

0997

0997

0993

Ornidazole

10ndash100

119910=867363119909+1629119910=578037119909+89570119910=381225119909+135625

0999

0999

0992

Oflo

xacin

10ndash100

119910=159216119909minus487671119910=622512119909+566425119910=512493119909+52237

0994

0990

0993

Ciprofl

oxacin

10ndash100

119910=799202119909minus207609119910=408103119909+42569119910=330771119909+255369

0996

0999

0996

Enroflo

xacin

10ndash100

119910=884074119909minus347422119910=691707119909+748835119910=429161119909+120416

0995

0998

0992


QAA1269 24-Nov-2016

1110-ZHHS-1TIC

849e327073

16946

386104

200 300 400 500 600 700 800100Time

0

100

()

1110-ZHHS-1TIC

545e3697187

22260 659

177454122

386104

644173

615165

723194

764205

200 300 400 500 600 700 800100Time

0

100

()

1110-ZHHS-1TIC

161e4739198

765205

200 300 400 500 600 700 800100Time

0

100

()

1110-ZHHS-1TIC

254e4672180

200 300 400 500 600 700 800100Time

0

100

()

1110-ZHHS-1TIC

889e3527141

389104

13436

200 300 400 500 600 700 800100Time

0

100

()

1110-ZHHS-1TIC

129e4647173

200 300 400 500 600 700 800100Time

0

100

()

















Metronidazole5 827 (0044) 810 (0058)25 857 (0031) 1007 (0050)50 861 (0020) 990 (0024)

Tinidazole5 1156 (0050) 8560 (0027)25 872 (0031) 1005 (0051)50 826 (0012) 825 (0039)

Ornidazole5 1133 (0044) 868 (0017)25 932 (0049) 1018 (0036)50 852 (0043) 1089 (0007)

Ofloxacin5 1167 (0046) 829 (0057)25 886 (0054) 948 (0039)50 819 (0019) 1065 (0023)

Ciprofloxacin5 831 (0027) 831 (0027)25 968 (0040) 968 (0040)50 1168 (0050) 818 (0033)

Enrofloxacin5 1099 (0053) 865 (0063)25 1159 (0044) 1039 (0062)50 1120 (0031) 1159 (0020)



4 Conclusion





Acknowledgments








References





































Journal of

Chemistry





Journal of

Volume 2018






Volume 2018




Journal of






Enzyme Research


Journal of



MaterialsJournal of






Na

nom

ate

ria

ls






0

20

40

60

80

100

120

Reco

very

()


0

20

40

60

80

100

120

Reco

very

()


0

20

40

60

80

100

120

Reco

very

()


0

20

40

60

80

100

120

Reco

very

()


20

40

60

80

100

120

Reco

very

()


0

20

40

60

80

100

120

Reco

very

()





QAA1269 24-Nov-2016

Time100 200 300 400 500 600 700 800


203e427073

0

100

()


143e4693186

24165

0124 379

1020

100

()

200 300 400 500 600 700 800100Time


260e4735197

0

100

()

200 300 400 500 600 700 800100Time


574e4672180

0

100

()

200 300 400 500 600 700 800100Time


132e4523140

0154

377101

23964

392105

0

100

()

200 300 400 500 600 700 800100Time


218e4647173

0

100(

)

200 300 400 500 600 700 800100Time

204638 24-Nov-2016







Table3Linear

rangeregressio

nequatio

nandcorrela

tioncoeffi

cients

Com

poun

dLinear

rang

e(ng

mL)

Calib

ratio

nequatio

nCorrelationcoeffi

cients1198772

Standard

solutio

nsAc

aciaho

ney

Jujube

honey

Standard

solutio

nsAc

aciaho

ney

Jujube

honey

Metronidazole

10ndash100

119910=149667119909+228847119910=842987119909+173078119910=366167119909+330998

0998

0997

0996

Tinidazole

10ndash100

119910=693581119909+468099119910=471843119909minus590018119910=335304119909+83281

0997

0997

0993

Ornidazole

10ndash100

119910=867363119909+1629119910=578037119909+89570119910=381225119909+135625

0999

0999

0992

Oflo

xacin

10ndash100

119910=159216119909minus487671119910=622512119909+566425119910=512493119909+52237

0994

0990

0993

Ciprofl

oxacin

10ndash100

119910=799202119909minus207609119910=408103119909+42569119910=330771119909+255369

0996

0999

0996

Enroflo

xacin

10ndash100

119910=884074119909minus347422119910=691707119909+748835119910=429161119909+120416

0995

0998

0992


QAA1269 24-Nov-2016

1110-ZHHS-1TIC

849e327073

16946

386104

200 300 400 500 600 700 800100Time

0

100

()

1110-ZHHS-1TIC

545e3697187

22260 659

177454122

386104

644173

615165

723194

764205

200 300 400 500 600 700 800100Time

0

100

()

1110-ZHHS-1TIC

161e4739198

765205

200 300 400 500 600 700 800100Time

0

100

()

1110-ZHHS-1TIC

254e4672180

200 300 400 500 600 700 800100Time

0

100

()

1110-ZHHS-1TIC

889e3527141

389104

13436

200 300 400 500 600 700 800100Time

0

100

()

1110-ZHHS-1TIC

129e4647173

200 300 400 500 600 700 800100Time

0

100

()

















Metronidazole5 827 (0044) 810 (0058)25 857 (0031) 1007 (0050)50 861 (0020) 990 (0024)

Tinidazole5 1156 (0050) 8560 (0027)25 872 (0031) 1005 (0051)50 826 (0012) 825 (0039)

Ornidazole5 1133 (0044) 868 (0017)25 932 (0049) 1018 (0036)50 852 (0043) 1089 (0007)

Ofloxacin5 1167 (0046) 829 (0057)25 886 (0054) 948 (0039)50 819 (0019) 1065 (0023)

Ciprofloxacin5 831 (0027) 831 (0027)25 968 (0040) 968 (0040)50 1168 (0050) 818 (0033)

Enrofloxacin5 1099 (0053) 865 (0063)25 1159 (0044) 1039 (0062)50 1120 (0031) 1159 (0020)



4 Conclusion





Acknowledgments








References





































Journal of

Chemistry





Journal of

Volume 2018






Volume 2018




Journal of






Enzyme Research


Journal of



MaterialsJournal of






Na

nom

ate

ria

ls





QAA1269 24-Nov-2016

Time100 200 300 400 500 600 700 800


203e427073

0

100

()


143e4693186

24165

0124 379

1020

100

()

200 300 400 500 600 700 800100Time


260e4735197

0

100

()

200 300 400 500 600 700 800100Time


574e4672180

0

100

()

200 300 400 500 600 700 800100Time


132e4523140

0154

377101

23964

392105

0

100

()

200 300 400 500 600 700 800100Time


218e4647173

0

100(

)

200 300 400 500 600 700 800100Time

204638 24-Nov-2016







Table3Linear

rangeregressio

nequatio

nandcorrela

tioncoeffi

cients

Com

poun

dLinear

rang

e(ng

mL)

Calib

ratio

nequatio

nCorrelationcoeffi

cients1198772

Standard

solutio

nsAc

aciaho

ney

Jujube

honey

Standard

solutio

nsAc

aciaho

ney

Jujube

honey

Metronidazole

10ndash100

119910=149667119909+228847119910=842987119909+173078119910=366167119909+330998

0998

0997

0996

Tinidazole

10ndash100

119910=693581119909+468099119910=471843119909minus590018119910=335304119909+83281

0997

0997

0993

Ornidazole

10ndash100

119910=867363119909+1629119910=578037119909+89570119910=381225119909+135625

0999

0999

0992

Oflo

xacin

10ndash100

119910=159216119909minus487671119910=622512119909+566425119910=512493119909+52237

0994

0990

0993

Ciprofl

oxacin

10ndash100

119910=799202119909minus207609119910=408103119909+42569119910=330771119909+255369

0996

0999

0996

Enroflo

xacin

10ndash100

119910=884074119909minus347422119910=691707119909+748835119910=429161119909+120416

0995

0998

0992


QAA1269 24-Nov-2016

1110-ZHHS-1TIC

849e327073

16946

386104

200 300 400 500 600 700 800100Time

0

100

()

1110-ZHHS-1TIC

545e3697187

22260 659

177454122

386104

644173

615165

723194

764205

200 300 400 500 600 700 800100Time

0

100

()

1110-ZHHS-1TIC

161e4739198

765205

200 300 400 500 600 700 800100Time

0

100

()

1110-ZHHS-1TIC

254e4672180

200 300 400 500 600 700 800100Time

0

100

()

1110-ZHHS-1TIC

889e3527141

389104

13436

200 300 400 500 600 700 800100Time

0

100

()

1110-ZHHS-1TIC

129e4647173

200 300 400 500 600 700 800100Time

0

100

()

















Metronidazole5 827 (0044) 810 (0058)25 857 (0031) 1007 (0050)50 861 (0020) 990 (0024)

Tinidazole5 1156 (0050) 8560 (0027)25 872 (0031) 1005 (0051)50 826 (0012) 825 (0039)

Ornidazole5 1133 (0044) 868 (0017)25 932 (0049) 1018 (0036)50 852 (0043) 1089 (0007)

Ofloxacin5 1167 (0046) 829 (0057)25 886 (0054) 948 (0039)50 819 (0019) 1065 (0023)

Ciprofloxacin5 831 (0027) 831 (0027)25 968 (0040) 968 (0040)50 1168 (0050) 818 (0033)

Enrofloxacin5 1099 (0053) 865 (0063)25 1159 (0044) 1039 (0062)50 1120 (0031) 1159 (0020)



4 Conclusion





Acknowledgments








References





































Journal of

Chemistry





Journal of

Volume 2018






Volume 2018




Journal of






Enzyme Research


Journal of



MaterialsJournal of






Na

nom

ate

ria

ls





Table3Linear

rangeregressio

nequatio

nandcorrela

tioncoeffi

cients

Com

poun

dLinear

rang

e(ng

mL)

Calib

ratio

nequatio

nCorrelationcoeffi

cients1198772

Standard

solutio

nsAc

aciaho

ney

Jujube

honey

Standard

solutio

nsAc

aciaho

ney

Jujube

honey

Metronidazole

10ndash100

119910=149667119909+228847119910=842987119909+173078119910=366167119909+330998

0998

0997

0996

Tinidazole

10ndash100

119910=693581119909+468099119910=471843119909minus590018119910=335304119909+83281

0997

0997

0993

Ornidazole

10ndash100

119910=867363119909+1629119910=578037119909+89570119910=381225119909+135625

0999

0999

0992

Oflo

xacin

10ndash100

119910=159216119909minus487671119910=622512119909+566425119910=512493119909+52237

0994

0990

0993

Ciprofl

oxacin

10ndash100

119910=799202119909minus207609119910=408103119909+42569119910=330771119909+255369

0996

0999

0996

Enroflo

xacin

10ndash100

119910=884074119909minus347422119910=691707119909+748835119910=429161119909+120416

0995

0998

0992


QAA1269 24-Nov-2016

1110-ZHHS-1TIC

849e327073

16946

386104

200 300 400 500 600 700 800100Time

0

100

()

1110-ZHHS-1TIC

545e3697187

22260 659

177454122

386104

644173

615165

723194

764205

200 300 400 500 600 700 800100Time

0

100

()

1110-ZHHS-1TIC

161e4739198

765205

200 300 400 500 600 700 800100Time

0

100

()

1110-ZHHS-1TIC

254e4672180

200 300 400 500 600 700 800100Time

0

100

()

1110-ZHHS-1TIC

889e3527141

389104

13436

200 300 400 500 600 700 800100Time

0

100

()

1110-ZHHS-1TIC

129e4647173

200 300 400 500 600 700 800100Time

0

100

()

















Metronidazole5 827 (0044) 810 (0058)25 857 (0031) 1007 (0050)50 861 (0020) 990 (0024)

Tinidazole5 1156 (0050) 8560 (0027)25 872 (0031) 1005 (0051)50 826 (0012) 825 (0039)

Ornidazole5 1133 (0044) 868 (0017)25 932 (0049) 1018 (0036)50 852 (0043) 1089 (0007)

Ofloxacin5 1167 (0046) 829 (0057)25 886 (0054) 948 (0039)50 819 (0019) 1065 (0023)

Ciprofloxacin5 831 (0027) 831 (0027)25 968 (0040) 968 (0040)50 1168 (0050) 818 (0033)

Enrofloxacin5 1099 (0053) 865 (0063)25 1159 (0044) 1039 (0062)50 1120 (0031) 1159 (0020)



4 Conclusion





Acknowledgments








References





































Journal of

Chemistry





Journal of

Volume 2018






Volume 2018




Journal of






Enzyme Research


Journal of



MaterialsJournal of






Na

nom

ate

ria

ls





QAA1269 24-Nov-2016

1110-ZHHS-1TIC

849e327073

16946

386104

200 300 400 500 600 700 800100Time

0

100

()

1110-ZHHS-1TIC

545e3697187

22260 659

177454122

386104

644173

615165

723194

764205

200 300 400 500 600 700 800100Time

0

100

()

1110-ZHHS-1TIC

161e4739198

765205

200 300 400 500 600 700 800100Time

0

100

()

1110-ZHHS-1TIC

254e4672180

200 300 400 500 600 700 800100Time

0

100

()

1110-ZHHS-1TIC

889e3527141

389104

13436

200 300 400 500 600 700 800100Time

0

100

()

1110-ZHHS-1TIC

129e4647173

200 300 400 500 600 700 800100Time

0

100

()

















Metronidazole5 827 (0044) 810 (0058)25 857 (0031) 1007 (0050)50 861 (0020) 990 (0024)

Tinidazole5 1156 (0050) 8560 (0027)25 872 (0031) 1005 (0051)50 826 (0012) 825 (0039)

Ornidazole5 1133 (0044) 868 (0017)25 932 (0049) 1018 (0036)50 852 (0043) 1089 (0007)

Ofloxacin5 1167 (0046) 829 (0057)25 886 (0054) 948 (0039)50 819 (0019) 1065 (0023)

Ciprofloxacin5 831 (0027) 831 (0027)25 968 (0040) 968 (0040)50 1168 (0050) 818 (0033)

Enrofloxacin5 1099 (0053) 865 (0063)25 1159 (0044) 1039 (0062)50 1120 (0031) 1159 (0020)



4 Conclusion





Acknowledgments








References





































Journal of

Chemistry





Journal of

Volume 2018






Volume 2018




Journal of






Enzyme Research


Journal of



MaterialsJournal of






Na

nom

ate

ria

ls










Metronidazole5 827 (0044) 810 (0058)25 857 (0031) 1007 (0050)50 861 (0020) 990 (0024)

Tinidazole5 1156 (0050) 8560 (0027)25 872 (0031) 1005 (0051)50 826 (0012) 825 (0039)

Ornidazole5 1133 (0044) 868 (0017)25 932 (0049) 1018 (0036)50 852 (0043) 1089 (0007)

Ofloxacin5 1167 (0046) 829 (0057)25 886 (0054) 948 (0039)50 819 (0019) 1065 (0023)

Ciprofloxacin5 831 (0027) 831 (0027)25 968 (0040) 968 (0040)50 1168 (0050) 818 (0033)

Enrofloxacin5 1099 (0053) 865 (0063)25 1159 (0044) 1039 (0062)50 1120 (0031) 1159 (0020)



4 Conclusion





Acknowledgments








References





































Journal of

Chemistry





Journal of

Volume 2018






Volume 2018




Journal of






Enzyme Research


Journal of



MaterialsJournal of






Na

nom

ate

ria

ls










References





































Journal of

Chemistry





Journal of

Volume 2018






Volume 2018




Journal of






Enzyme Research


Journal of



MaterialsJournal of






Na

nom

ate

ria

ls






References





































Journal of

Chemistry





Journal of

Volume 2018






Volume 2018




Journal of






Enzyme Research


Journal of



MaterialsJournal of






Na

nom

ate

ria

ls














Journal of

Chemistry





Journal of

Volume 2018






Volume 2018




Journal of






Enzyme Research


Journal of



MaterialsJournal of






Na

nom

ate

ria

ls









Journal of

Chemistry





Journal of

Volume 2018






Volume 2018




Journal of






Enzyme Research


Journal of



MaterialsJournal of






Na

nom

ate

ria

ls




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