Development of a simple liquid chromatography-tandem mass spectrometry method for multiresidue...

1650emsp Yu et al Journal of aoaC InternatIonal Vol 94 no 5 2011

DevelopmentemspofemspaemspSimpleemspLiquidemspChromatography-TandememspMassemspSpectrometryemspMethodemspforemspMultiresidueemspDeterminationemspofemspAntifungalemspDrugsemspinemspChickenemspTissuesYingemspYuBeijing Center for Disease Control and Prevention Beijing 100013 Peoplersquos Republic of China Beijing University of Chemical Technology College of Science Institute of Applied Chemistry Beijing 100029 Peoplersquos Republic of ChinaJingemspZhangBeijing Center for Disease Control and Prevention Beijing 100013 Peoplersquos Republic of ChinaBingemspShao

1

Beijing Center for Disease Control and Prevention Beijing 100013 Peoplersquos Republic of China Nanchang University School of Life Sciences and Food Engineering Nanchang 330031 Peoplersquos Republic of ChinaYongningemspWuChina Center for Disease Control and Prevention Institute of Nutrition and Food Safety Beijing 100085 Peoplersquos Republic of ChinaHejunemspDuanBeijing Center for Disease Control and Prevention Beijing 100013 Peoplersquos Republic of Chinahongtao LiuBeijing University of Chemical Technology College of Science Institute of Applied Chemistry Beijing 100029 Peoplersquos Republic of China

Received June 23 2010 Accepted by JB February 10 20111 Corresponding authorrsquos e-mail shaobingchsinacomDOI 105740jaoacint10-244

VETERINARY DRUG RESIDUES

A method involving LC coupled with MSMS (LCMSMS) was designed for simultaneous quantification of 10 antifungal drugs (voriconazole griseofulvin clotrimazole bifonazole econazole ketoconazole itraconazole miconazole terconazole and fluconazole) in the liver and muscles of chickens Homogenized tissue samples were extracted with acetonitrile and subsequently underwent freezing-delipidation A Waters Acquity Ultra Performance LC BEH C18 column was used to separate the analytes coupled with MSMS using an electrospray ionization source The accuracy of the method was confirmed with a mean recovery of 71ndash121 and acceptable coefficients of variation (4ndash23 n = 6) The detection capability of these compounds in two different matrixes was 050ndash282 microgkg This method can be applied for the screening and confirmation of target antifungal drugs in chicken tissues

Livestock husbandry is associated with various pharmaceuticals used on a large scale to prevent or control microbial infection increase meat

production and reduce cost One of the most important classes of drugs is the antifungal agent group commonly used in veterinary husbandry to treat various diseases caused by Aspergillus fumigatus Candida albicans and Cryptococcus neoformans (1) Based on their chemical structure antifungal drugs can be classified as polyene antibiotics (eg amphotericin B nystatin) fluorinated pyrimidine (5-fluorocytosine) azoles (miconazole terconazole and fluconazole) and allylamine derivatives (griseofulvin)

Azoles and griseofulvin are widely applied for treating avian mycoses (2) Their inappropriate or illicit administration in veterinary husbandry may lead to high-level residues of these drugs in edible tissues It has been reported that azoles can induce adverse effects due to the inhibition of steroid synthesis in mammals (3) whereas griseofulvin is neurotoxic hepatotoxic and carcinogenic (4) Antifungal resistance is also a growing concern in birds and mammals including humans (5 6) In view of such potential harm to the health of consumers sample detection and market surveillance are therefore important

There are many LC-based methods for determination of antifungal drugs in biosamples including LC coupled with UV (4ndash20) or fluorescence detection (3 21) and the LCMS technique (22 23) Most of these methods can be used to reliably quantify one or two drugs in plasma or serum (3 7ndash12 21) In the past few years antifungal drugs have presented additional environmental and human safety concerns because pharmaceuticals and personal care products are increasingly being discarded

Yu et al Journal of aoaC InternatIonal Vol 94 no 5 2011 1651

into the environment (24ndash26) An LCMS method that can simultaneously detect nine drug residues including three antifungals in wastewater was reported recently (25) Analytical methods for the determination of nystatin in rabbit tissues and amphotericin B in chicken livers were established using LC-UV by Groll et al (19) and Echevarriacutea et al (20) respectively However few studies have focused on the analysis of residues of azoles in food of animal origin Azole antifungal drugs are not listed in European Regulation 372010EU (27) or in the US Food and Drug Administration Code of Federal Regulations Title 21 (28) Many azoles are frequently used in poultry and other birds (2 29) however and clotrimazole miconazole and griseofulvin are authorized for veterinary purposes in China (30) Many antifungal drugs also might be used illicitly during poultry breeding so developing a comprehensive detection method is necessary for surveillance

The aim of the present study was to develop a comprehensive method for the simultaneous determination of 10 antifungal drugs (including azoles and griseofulvin) in the liver and muscles of chickens

Experimental

Apparatus

(a) HomogenizermdashB-400 (Buumlchi Postfach Flawil Switzerland)

(b) Electronic balancemdashBS110S (Sartorius Inc Goettingen Lower Sachsen Germany)

(c) Centrifuge tubesmdash50 mL (Code 2343-050 Asahi Glass Co Ltd Tokyo Japan)

(d) Centrifuge tubesmdash15 mL (MCT-150-C Axygen Scientific Union City CA)

(e) Glass vialsmdash40 mL (La-Pha-Pack Langerwerde Germany)

(f) Vortex mixermdashSA8 (Bibby Sterilin Ltd Stone UK)(g) CentrifugemdashAllegratrade X-22R (Beckman Inc

Fullerton CA)(h) Nitrogen evaporatormdashN-Evaptrade 116 (Organomation

Associates Inc Berlin MA)(i) LC systemmdashAcquitytrade-Ultra Performance LC

(Waters Milford MA)(j) MSMS systemmdashMicromass Quattro Ultimatrade Pt

controlled by MassLynx Ver 41 software (Manchester New Hampshire UK)

(k) LC columnmdashBEH C18 21 times 100 mm 17 μm (Waters)

(l) Water purification systemmdashMillipore-Elix-QE-QG (Millipore Inc Billerica MA)

(m) Muffle furnacemdashL 911B 170 (Nabertherm Industrial Furnaces Ltd Lilienthal Bremen Germany)

Reagents

(a) MethanolmdashHPLC grade (Dima Technology Inc Richmond Hill VA)

(b) AcetonitrilemdashHPLC grade (Dima Technology Inc)

(c) AcetonemdashHPLC grade (Dima Technology Inc)

Table 1 Instrument conditions for antifungal drug analyses Fluconazole Other 9 analytes

LC conditions Mobile phase A Water A Water

B Acetonitrile B Methanol

Gradient list Time (min) 0 25 26 45 46 Time (min) 0 40 80 81

Total flow 03 mLmin

Column Acquity UPLC BEH C18 column (100 times 21 mm 17 μm)

MS conditions Ionization mode ESIndash ESI+

Capillary voltage 30 kV 22 kV

Desolvation gas 550 Lh

Source temperature 100degC

Desolvation gas temperature 350degC

Multiplier voltage 650 V

RF lens 1 24 28

Aperture 05 08

RF lens 2 05 05

Collision gas Ultra-high purity argon

Pressure of collision chamber 32 times 10ndash3 mbar

Entrance voltage ndash1

Exit voltage 2


(d) Ultra-pure watermdashMade by a Milli-Q Water Purification System (Millipore Corp Bedford MA)

(e) Sodium sulfate (anhydrous)mdashAnalytical grade (Chemical Reagent Beijing Co Ltd Beijing China) Baked at 600degC for approximately 3 h in the muffle furnace before use

(f) StandardsmdashVoriconazole [purity 995 Toronto Research Chemicals (TRC) Inc North York ON Canada] griseofulvin (purity gt95 SigmandashAldrich St Louis MO) clotrimazole [purity 99 National Institute for the Control of Pharmaceutical and Biological Products (NICPBP) Beijing China] bifonazole (purity gt98 NICPBP) econazole (purity 990 Dr Ehrenstorfer GmbH Augsburg Bayern Germany) ketoconazole (purity 97 NICPBP) miconazoleemsp(purity 99 NICPBP) itraconazole (purity 99 NICPBP) terconazole [purity 1000 United States Pharmacopeia (USP) Rockville MD] and fluconazole (purity 1000 USP)

(g) Isotope-labeled internal standards (IS)mdashAll the isotope-labeled standards were purchased from TRC voriconazole-d3 (purity 995) clotrimazole-d5 (purity 995) ketoconazole-d8 (purity 995) itraconazole-d5 (purity 995) fluconazole-d4 (purity 995)

Preparation of Standard Solutions

(a) Stock standard solutionsmdashIndividual stock standard solution (1 mgmL) was prepared by dissolving 10 mg of each analyte in 10 mL methanol as well as dissolving 1 mg of the isotope-labeled IS in 1 mL methanol

(b) Intermediate stock standardmdashThe intermediate stock standard was 10 mgL made by adding 100 microL of each stock solution to an individual 10 mL volume flask and diluting to the required volume with methanol

(c) Working mixed standard solutionsmdashWorking solutions at serial concentrations (10 20 50 100 200 and 500 microgL) were obtained by combining aliquots of intermediate stock solutions followed by subsequent dilution with methanol

(d) Working IS solutionmdashThe working IS solution was 100 microgL made by combining 100 microL of each intermediate stock solution of the IS in an individual 10 mL flask and diluting to the required volume with methanol

All the standard solutions were stored in brown glass

Table 2 LCMSMS acquisition parameters for the determination of antifungal drugs

Analyte Transition Cone voltage V Collision energy eV Retention time min Internal standard

Griseofulvin 3530 to 1651a

3530 to 285144 17

17235 plusmn 003 Voriconazole-d3

Voriconazole 3501 to 2811a

3501 to 127235 14


Voriconazole-d3 3529 to 2839 35 14 253 plusmn 002

Ketoconazole 5311 to 1121a

5311 to 82450 36

36366 plusmn 002 Ketoconazole-d8

Ketoconazole-d8 5389 to 1191 50 36 365 plusmn 004

Clotrimazole 2777 to 1663a

2777 to 242240 16

18394 plusmn 002 Clotrimazole-d5

Clotrimazole-d5 2827 to 1694 40 16 395 plusmn 002

Bifonazole 3111 to 2431a

3111 to 228048 10

29395 plusmn 002 mdash

Itraconazole 7053 to 3920a

7053 to 432058 32

31404 plusmn 002 Itraconazole-d5

Itraconazole-d5 7109 to 3970 58 32 404 plusmn 002

Econazole 3813 to 1251a

3813 to 193335 22


Miconazole 4171 to 1609a

4171 to 229061 24


Terconazole 5319 to 2192a

5319 to 277139 30


Fluconazole 3051 to 1910a

3051 to 129935 11

20176 plusmn 001 Fluconazole-d4

Fluconazole-d4 3089 to 1940 35 11 176 plusmn 001

a Ion was used for quantification


bottles Stock standards were stable for ge6 months at ndash20degC whereas working solutions were prepared every week

Sample Preparation Extraction and Cleanup

Samples of liver and muscle from chickens were bought in Beijing supermarkets verified as being free of drugs

and used as blank samples for method development and validation

Tissue samples were homogenized Aliquots of 20 g samples were transferred into 50 mL centrifuge tubes and spiked with 200 microL working IS solution For the recovery test and preparation of matrix-fortified calibration standards an appropriate amount of working

Figure 1 LCMSMS chromatograms of antifungal drugs in a spiked chicken liver sample (spiking level 10 μgkg left) and a blank sample (right)


mixed standard was spiked An appropriate amount of anhydrous sodium sulfate (approximately 6ndash10 g) was added until free-flowing powder could be obtained after vortex mixing A 10 mL volume of acetonitrile was added to the mixture which was vortex-mixed for 2 min and then sonicated for 10 min at 40degC After cooling to room temperature the mixture was centrifuged at 10 000 rpm (7286 times g) for 10 min at 4degC The supernatant was decanted into a 40 mL glass vial and residues were extracted again with 10 mL acetonitrile The supernatants were combined and evaporated to dryness under a gentle stream of nitrogen at 40degC The residues were reconstituted with 1 mL methanol containing 01 (vv) formic acid The resulting solution was transferred into

a 15 mL Eppendorf tube and stored at ndash20degC overnight It was then centrifuged at 15 000 rpm for 5 min at 4degC aliquots of the supernatant were injected into the LCMSMS system

LCMSMS Analyses

Liquid chromatographic separation was done on a Waters Acquity Ultra Performance LC system based on separation by an Acquity BEH C18 column (21 times 100 mm 17 μm) at 40degC The injection volume was 5 μL Sample extracts were analyzed by a Micromass Quattro Ultima Pt Mass Spectrometer equipped with an electrospray ionization (ESI) interface in selected reaction monitoring

Table 3 Signal suppressiona of analytes in chicken liver extracts using different treatment procedures

Analyte Acetonitrile extracted Acetonitrile extraction followed by

purification using an NH2 cartridge Acetonitrile extraction followed by

freezing filtration

Griseofulvin 77 0 0

Voriconazole 74 0 45

Ketoconazole 79 36 0

Clotrimazole 76 0 49

Bifonazole 98 57 86

Itraconazole 95 88 75

Econazole 95 75 83

Miconazole 94 91 56

Terconazole 96 29 68

Fluconazole 86 10 0

a Signal suppression () = (1 ndash SmS0) times 100 where Sm is the slope of the matrix-matched standard curve and S0 is the slope of the pure standard curve

Table 4 Slopes of neat standard calibration curves and different matrix-fortified calibration curves

Chicken muscle Chicken liver

Analyte Internal standard Standard S1a S2 S3 S1 S2 S3 RSD

Griseofulvin Voriconazole-d3 0224 0351 0225 0297 0259 0253 0352 19

Voriconazole Voriconazole-d3 0733 0843 0663 0759 0789 0755 0852 8

Ketoconazole Ketoconazole-d8 0309 0331 0324 0376 023 0321 0319 14

Clotrimazole Clotrimazole-d5 0855 0729 0637 0838 0767 0826 0851 10

Bifonazole Clotrimazole-d5 5539 3879 5277 8894 10548 5884 5118 37

Itraconazole-d5 8125 15099 21745 13531 24701 12143 13917 37

Itraconazole Itraconazole-d5 1287 1528 1233 1479 1234 1618 1774 14

Econazole Clotrimazole-d5 29648 14621 24883 20587 28876 35149 16388 31

Itraconazole-d5 70968 50873 10193 93354 75968 72111 33241 33

Miconazole Clotrimazole-d5 5762 7374 1897 5169 13334 17328 19128 66

Itraconazole-d5 14575 22677 7981 14498 21126 35129 30530 46

Terconazole Ketoconazole-d8 20715 22056 26643 27096 26129 25179 32975 16

Fluconazole Fluconazole-d4 0187 0170 0152 0163 0171 0168 0169 6a S1 S2 and S3 indicate different tissue samples


mode Nitrogen (purity 999) was the desolvation gas LC and MS parameters are summarized in Table 1 The compound-dependent cone voltage and collision energy are shown in Table 2

Calculations

Calibration curves for the target compounds (except for bifonazole miconazole and econazole) were obtained by carrying out a linear regression analysis on the ratio of standard solution areas to IS areas versus analyte concentrations from 1 to 50 μgL with 20 μgL IS Quantification of bifonazole miconazole and econazole was based on matrix-fortified curves ranging from 1 to 25 μgkg

Evaluation of the matrix effect was conducted using the strategy applied by Matuszewski et al (31) with slight modification Signal suppression was defined as 1 minus the ratio between the slope of the matrix-matched standard curves and the slope of the standard-solution curves then multiplied by 100 to obtain a percentage Absolute recoveries were used to evaluate the efficiency of sample preparation and were calculated as the slope of matrix-fortified standard curves divided by the slopes of matrix-matched standard curves and then multiplied by 100 to obtain a percentage

According to the literature the zero-tolerance principle could be applied for drugs not subjected to legislation regarding maximum residue limits (32ndash34) According to European Commission Decision 2002657EC (35) the decision limit (CCα) and detection capability (CCβ) for nonpermitted substances were determined as follows

CCα = 3SN20 representative blank samples

analyzing 20 blank tissue samples per matrix to be able to

calculate the SN at the time window in which the analyte is expected then 3 times the SN to be used as CCα

CCβ = CCα + 164 SD20 representative samples

spiked at CCα level

Twenty control blank samples (liver and muscle) were analyzed to determine the specificity of the method by looking for interfering peaks within a 25 margin of the relative retention time of each compound

Results and Discussion

LCMSMS

Following the gradient profile shown in Table 1 the mobile phase compositions (ie waterndashmethanol waterndashacetonitrile water containing 01 formic acidndashmethanol and water containing 01 formic acidndashacetonitrile) were optimized to achieve maximal sensitivity Results indicated that higher sensitivity and good chromatographic behavior could be achieved under the ESI positive mode if waterndashmethanol was used Waterndashacetonitrile was more desirable for the ESI negative mode

MS tuning of the various analytes was done by flow-injecting a standard into the mass spectrometer [M+H]+ was the most abundant ion for most analytes except fluconazole and clotrimazole [M-H]ndash was found to be the dominant ion for fluconazole The most prominent ion of clotrimazole was mz 2777 which originated from the in-source collision-induced dissociation and corresponded to [M-C3H2N2]

+ These ions were chosen as precursor ions product ions were subsequently optimized (Table 2) The boldfaced transition stated in Table 2 is selected for quantification whereas the second less-sensitive one is used for confirmation For the IS only one transition was monitored Chromatograms of target compounds are given in Figure 1 which shows the reconstructed ion chromatograms of a blank sample and a spiked sample of chicken liver This indicated good selectivity because there were no interferences at the elution time of the target analytes

Sample Preparation

The chicken liver matrix was used as the typical case for the development of the pretreatment method because of its complexity Octanolndashwater partition coefficients (logKow calculated by KOWWINTM in EPI SuiteTM software (Ver 311 US Environmental Protection Agency httpwwwepagovexposurepubsepisuitehtm) for the 10 target drugs ranged from 058 (fluconazole) to 586 (miconazole) demonstrating a wide range of polarity Methanol acetonitrile and acetone thus were initially used as extractants for screening purposes Ten milliliters

Table 5 CCα and CCβ of the analytes in samples of chicken liver and chicken muscle

Analyte

Chicken liver Chicken muscle

CCα microgkg

CCβ microgkg

CCα microgkg

CCβ microgkg

Griseofulvin 058 232 156 282

Voriconazole 045 125 026 068

Ketoconazole 051 144 047 107

Clotrimazole 014 211 046 161

Bifonazole 005 095 014 079

Itraconazole 094 210 076 183

Econazole 007 110 010 050

Miconazole 016 111 028 082

Terconazole 054 210 112 245

Fluconazole 033 141 034 089


of acetonitrile methanol or acetone were added to 2 g aliquots of chicken liver with analytes spiked at 10 μgkg As a result average recoveries for most target compounds at this concentration for a single extraction were 76ndash95 55ndash93 and 14ndash84 for acetonitrile methanol and acetone respectively A second extraction with 10 mL acetonitrile was found to increase the recovery by 10 while a third 10 mL extraction did not result in any significant improvement in recovery (lt3) As a result two 10 mL extractions were performed with acetonitrile for an optimized recovery of the target analytes from chicken liver

Purification procedures including liquidndashliquid extraction by hexane and SPE steps were evaluated to minimize the interference of matrixes such as lipids Hexane has been commonly used for lipid removal during biosample analyses Here hexane purification was first evaluated using a standard solution 1 mL hexane was added to 1 mL acetonitrilendashdissolved mixed standard (20 microgL) then vortex-mixed vigorously and centrifuged at 8000 rpm for 1 min The acetonitrile and hexane layers (the latter dried under a gentle stream of nitrogen and reconstituted with 1 mL acetonitrile) were detected by LCMSMS The results indicated that gt50 of analytes such as itraconazole clotrimazole and miconazole were extracted into hexane due to their low polarity We also carried out this experiment using fortified chicken liver extracts The result was similar to that of the pure standard solution thus hexane was not suitable for the purification procedure

SPE is frequently applied for concentration and purification in the detection of trace-level analytes in complex matrixes (including food and environmental samples) In the present study two RP SPE cartridges (C18 and HLB) and a normal-phase NH2 SPE cartridge were examined with respect to the matrix effect and recovery For the former two cartridges the condition solution was 6 mL methanol and 6 mL water the eluting solution was 6 mL methanol As well as for the NH2 cartridge 6 mL methanol and 6 mL acetonitrile were used as the condition and elution solutions respectively The final extracts looked cleaner after SPE however several analytes such as fluconazole and terconazole were weakly retained on the C18 and HLB cartridges and resulted in poor recoveries (lt50) Acceptable recoveries (71ndash121) were observed for the NH2 cartridge Nevertheless ion suppression of some target compounds (eg itraconazole econazole and miconazole) was very high (gt70 Table 3)

Freezing delipidation has been found to be an effective process to eliminate triacylglycerols and phospholipids which are major lipids in meat tissue (36) This approach was assessed in the present study according to the difference in freezing points between lipids and antifungal drugs After being frozen overnight at ndash20degC the final extract solution exhibited significantly lower ion signal suppression with virtually no change in the recoveries of target compounds In view of its simplicity and ability to provide a cleaner extract the freezing-defattening step was included in the procedure despite the fact that it significantly increases the time for sample preparation

Method Validation

Isotope dilution is a favorable method to compensate for the loss of target compounds in sample preparation and the ion suppression of the mass spectrometer However because it is difficult to obtain all isotopic standards for the

Table 6 Accuracy and precision of 10 target compounds from fortified processed chicken liver and chicken muscle

Analyte

Spiking level microgkg


Accuracy

Precision

Accuracy

Precision

Griseofulvin 3 121 23 112 20

6 107 17 110 22

12 104 16 106 19

Voriconazole 15 98 18 109 21

3 93 17 105 20

6 89 15 99 8

Ketocon-azole

15 104 19 98 16

3 106 20 93 14

6 114 18 107 14

Clotrimazole 3 113 15 88 13

6 99 15 90 12

12 120 14 91 4

Bifonazole 15 71 14 88 20

3 88 13 76 8

6 90 12 89 9

Itraconazole 3 82 12 95 17

6 97 12 108 14

12 101 11 117 7

Econazole 15 84 15 109 21

3 78 10 98 18

6 77 9 115 15

Miconazole 15 71 13 76 14

3 82 12 78 14

6 88 9 87 13

Terconazole 3 91 15 112 20

6 90 11 106 8

12 102 11 113 10

Fluconazole

15 91 8 92 9

3 105 5 105 9

6 106 5 96 6


target drugs in multiresidual analyses several compounds were sometimes analyzed with one isotopic standard The standard curves in methanol and in different matrixes were compared with appropriately screened ISs The slopes (Table 4) of most analytes in methanol and in different matrixes after cleanup were comparable RSDs le19 for seven of 10 compounds indicating that the choice of ISs was appropriate The ISs for bifonazole econazole and miconazole were not suitable because the RSDs of slopes were gt30 Thus matrix-fortified curves were used to quantify these three drugs

Good linearity was obtained for all analytes with correlation coefficients of r gt0995 CCα and CCβ of the analytes using this method were determined as described in the Experimental section and ranged from 007 to 156 microgkg and from 050 to 282 microgkg respectively (Table 5)

The accuracy of the method was confirmed with a mean recovery evaluated by the spiked blank samples at three concentrations with each condition carried out in six replicates Five tissue pools were used in the test The accuracy of the procedure ranged between 71 and 121 (Table 6) and the precision was represented by the RSD percentage at each fortification level for each compound these values summarized in Table 6 were lt23

The intraday and interday precisions were evaluated by spiking different samples of chicken liver at concentrations of 3 and 6 μgkg in five replicates within 1 day and over the course of 5 consecutive days respectively The intraday precision ranged from 5 to 19 and the interday precision ranged from 6 to 21 (Table 7) These results demonstrate the good reproducibility of the method

The robustness of the method was determined by analyzing the same samples under various conditions extraction temperatures (40 50 and 60degC) evaporating temperatures (25 30 35 40 and 45degC) and freezing time at ndash20degC (1 2 4 8 12 and 16 h) were compared As a result an extraction temperature of 40ndash50degC an

evaporating temperature between 25 and 40degC and freezing time between 4 and 16 h (overnight) did not have a significant effect The main advantages of the method are its relatively high selectivity and high sensitivity due to the LCMSMS detection system and ISs for quantification which make the method more accurate and precise

Conclusions

A simple sensitive and comprehensive analytical method based on liquid extraction followed by LCMSMS analyses was developed for simultaneous determination of 10 antifungal drugs in chicken tissues and can be used in the routine analysis of samples of chicken muscle and chicken liver

Acknowledgments

We gratefully acknowledge financial support from the National Key Technology Research amp Development Program (Grant No 2006BAK02A27) and the Key Projects of the Scientific Olympics in the Beijing Science amp Technology Special Program (Z07001000560708)

References

(1) Odds FC Brown AJP amp Gow NAR (2003) Trends Microbiol 11 272ndash279 doi101016S0966-842X(03)00117-3

(2) Orosz SE (2003) Vet Clin North Am Exot Anim Pract 6 337ndash350 doi101016S1094-9194(03)00008-2

(3) Srivatsan V Dasgupta AK Kale P Datla RR Soni D Patel M Patel R amp Mavadhiya C (2004) J Chromatogr A 1031 307ndash313 doi101016jchroma200311061

(4) Knasmuumlller S Parzefall W Helma C Kassie F Ecker S amp Schulte-Hermann R (1997) Crit Rev Toxicol 27 495ndash537 doi10310910408449709078444

Table 7 Intraday and interday reproducibility of 10 antifungal drugs in chicken liver

Analyte

Spiking level 30 microgkg Spiking level 60 microgkg

Intraday precision Interday precision Intraday precision Interday precision





Bifonazole 15 20 14 15


Econazole 13 19 13 15

Miconazole 16 20 11 13


Fluconazole 5 7 5 6


(5) Bhanderi BB Yadav MM amp Roy A (2009) Veterinary World 2 204ndash207

(6) Kolaczkowska A Kolaczkowska M Sokolowska A Miecznikowska H Kubiak A Rolka K amp Polanowski A (2010) Mycoses 53 314ndash320

(7) Chhun S Rey E Tran A Lortholary O Pons G amp Jullien V (2007) J Chromatogr B 852 223ndash228 doi101016jjchromb200701021

(8) Pennick GJ Clark M Sutton DA amp Rinaldi MG (2003) Antimicrob Agents Chemother 47 2348ndash2350 doi101128AAC4772348-23502003

(9) Kim SS Im HT Kang IM Lee HS Lee HW Cho SH Kim JB amp Lee KT (2007) J Chromatogr B 852 174ndash179 doi101016jjchromb200701012

(10) Puranajoti P Kasina R amp Tenjarla S (1999) J Clin Pharm Ther 24 445ndash450 doi101046j1365-2710199900251x

(11) Langman LJ amp Boakye-Agyeman F (2007) Clin Biochememsp40 1378ndash1385 doi101016jclinbiochem200707024

(12) Baranowska I Markowski P amp Baranowski J (2006) Anal Chim Acta 570 46ndash58 doi101016jaca200604002

(13) Hajkova R Sklenarova H Matysova L Svecova P amp Solich P (2007) Talanta 73 483ndash489 doi101016jtalanta200704023

(14) Abdel-Moety EM Khattab FI Kelani KM amp AbouAl-Alamein AM (2002) Il Farmaco 57 931ndash938 doi101016S0014-827X(02)01270-3

(15) Srinubabu G Raju Ch A Sarath N Kumar PK amp Rao JV (2007) Talanta 71 1424ndash1429 doi101016jtalanta200604042

(16) Akay C Ozkan SA Senturk Z amp Cevheroglu S (2002) Il Farmaco 57 953ndash957 doi101016S0014-827X(02)01296-X

(17) Vertzon MV Reppas C amp Archontaki HA (2006) J Chromatogr B 839 62ndash67 doi101016jjchromb200603010

(18) Kolpin DW Skopec M Meyer MT Furlong ET amp Zaugg SD (2004) Sci Total Environ 328 119ndash130 doi101016jscitotenv200401015

(19) Groll AH Mickiene D Werner K Piscitelli SC amp Walsh TJ (1999) J Chromatogr B 735 51ndash62 doi101016S0378-4347(99)00396-5

(20) Echevarriacutea I Barturen C Renedo MJ amp Dios-Vieacuteitez MC (1998) J Chromatogr A 819 171ndash176 doi101016S0021-9673(98)00425-7

(21) Michael C Teichert J amp Preiss R (2008)

J Chromatogr B 865 74ndash80 doi101016jjchromb200802009

(22) Zhou L Glickman RD Chen N Sponsel WE Graybill JR amp Lam KW (2002) J Chromatogr B 776 213ndash220 doi101016S1570-0232(02)00344-6

(23) Egle H Trittler R Konig A amp Kummerer K (2005) JChromatogr B 814 361ndash367 doi101016jjchromb200410069

(24) Roberts PH amp Thomas KV (2006) Sci Total Environ 356 143ndash153 doi101016jscitotenv200504031

(25) Van De Steene JC amp Lambert WE (2008) J Chromatogr A 1182 153ndash160 doi101016jchroma200801012

(26) Chen YL Felder L Jiang X amp Naidong W (2002) J Chromatogr B 774 67ndash78 doi101016S1570-0232(02)00209-X

(27) Commission Regulation 372010EU (2010) Off J Eur Commun 2012010 L170

(28) US Food and Drug Administration (1996) Fed Regist Title 21 CFR 201105

(29) Rochette F Engelen M amp Vanden Bossche H (2003) J Vet Pharmacol Ther 26 31ndash53 doi101046j1365-2885200300457x

(30) MZ Zhu (Ed) (2002) in Veterinary Drug Handbook Chinese Chemical Industry Press Beijing China pp 114ndash129

(31) Matuszewski BK Constanzer ML amp Chavez-Eng CM (2003) Anal Chem 75 3019ndash3030 doi101021ac020361s

(32) Mortier L Huet AC Daeseleire E Huyghebaert G Fodey T Elliott C Delahaut P amp Peteghem CV (2005) J Agric Food Chem 53 7142ndash7149 doi101021jf050836r

(33) Mortier L Daeseleire E amp Peteghem CV (2005) Rapid Commun Mass Spectrom 19 533ndash539 doi101002rcm1819

(34) Mortiera L Daeseleirea E amp Peteghemb CV (2005) J Chromatogr B 820 261ndash270 doi101016jjchromb200504016

(35) Commission Decision 2002657EC (2002) Off J Eur Commun Sec 3125 and 3126

(36) Seo J Kima H Chung BC amp Hong J (2005) J Chromatogr A 1067 303ndash309 doi101016jchroma200412063


into the environment (24ndash26) An LCMS method that can simultaneously detect nine drug residues including three antifungals in wastewater was reported recently (25) Analytical methods for the determination of nystatin in rabbit tissues and amphotericin B in chicken livers were established using LC-UV by Groll et al (19) and Echevarriacutea et al (20) respectively However few studies have focused on the analysis of residues of azoles in food of animal origin Azole antifungal drugs are not listed in European Regulation 372010EU (27) or in the US Food and Drug Administration Code of Federal Regulations Title 21 (28) Many azoles are frequently used in poultry and other birds (2 29) however and clotrimazole miconazole and griseofulvin are authorized for veterinary purposes in China (30) Many antifungal drugs also might be used illicitly during poultry breeding so developing a comprehensive detection method is necessary for surveillance

The aim of the present study was to develop a comprehensive method for the simultaneous determination of 10 antifungal drugs (including azoles and griseofulvin) in the liver and muscles of chickens

Experimental

Apparatus

(a) HomogenizermdashB-400 (Buumlchi Postfach Flawil Switzerland)

(b) Electronic balancemdashBS110S (Sartorius Inc Goettingen Lower Sachsen Germany)

(c) Centrifuge tubesmdash50 mL (Code 2343-050 Asahi Glass Co Ltd Tokyo Japan)

(d) Centrifuge tubesmdash15 mL (MCT-150-C Axygen Scientific Union City CA)

(e) Glass vialsmdash40 mL (La-Pha-Pack Langerwerde Germany)

(f) Vortex mixermdashSA8 (Bibby Sterilin Ltd Stone UK)(g) CentrifugemdashAllegratrade X-22R (Beckman Inc

Fullerton CA)(h) Nitrogen evaporatormdashN-Evaptrade 116 (Organomation

Associates Inc Berlin MA)(i) LC systemmdashAcquitytrade-Ultra Performance LC

(Waters Milford MA)(j) MSMS systemmdashMicromass Quattro Ultimatrade Pt

controlled by MassLynx Ver 41 software (Manchester New Hampshire UK)

(k) LC columnmdashBEH C18 21 times 100 mm 17 μm (Waters)

(l) Water purification systemmdashMillipore-Elix-QE-QG (Millipore Inc Billerica MA)

(m) Muffle furnacemdashL 911B 170 (Nabertherm Industrial Furnaces Ltd Lilienthal Bremen Germany)

Reagents

(a) MethanolmdashHPLC grade (Dima Technology Inc Richmond Hill VA)

(b) AcetonitrilemdashHPLC grade (Dima Technology Inc)

(c) AcetonemdashHPLC grade (Dima Technology Inc)

Table 1 Instrument conditions for antifungal drug analyses Fluconazole Other 9 analytes

LC conditions Mobile phase A Water A Water

B Acetonitrile B Methanol

Gradient list Time (min) 0 25 26 45 46 Time (min) 0 40 80 81

Total flow 03 mLmin

Column Acquity UPLC BEH C18 column (100 times 21 mm 17 μm)

MS conditions Ionization mode ESIndash ESI+

Capillary voltage 30 kV 22 kV

Desolvation gas 550 Lh

Source temperature 100degC

Desolvation gas temperature 350degC

Multiplier voltage 650 V

RF lens 1 24 28

Aperture 05 08

RF lens 2 05 05

Collision gas Ultra-high purity argon

Pressure of collision chamber 32 times 10ndash3 mbar

Entrance voltage ndash1

Exit voltage 2















3530 to 285144 17



3501 to 127235 14




5311 to 82450 36




2777 to 242240 16




3111 to 228048 10



7053 to 432058 32




3813 to 193335 22



4171 to 229061 24



5319 to 277139 30



3051 to 129935 11














LCMSMS Analyses





freezing filtration

Griseofulvin 77 0 0




Bifonazole 98 57 86


Econazole 95 75 83

Miconazole 94 91 56


Fluconazole 86 10 0










Itraconazole-d5 8125 15099 21745 13531 24701 12143 13917 37



Itraconazole-d5 70968 50873 10193 93354 75968 72111 33241 33


Itraconazole-d5 14575 22677 7981 14498 21126 35129 30530 46





Calculations











LCMSMS




Sample Preparation



Analyte


CCα microgkg

CCβ microgkg

CCα microgkg

CCβ microgkg





Bifonazole 005 095 014 079


Econazole 007 110 010 050

Miconazole 016 111 028 082








Method Validation



Analyte



Accuracy

Precision

Accuracy

Precision


6 107 17 110 22

12 104 16 106 19


3 93 17 105 20

6 89 15 99 8

Ketocon-azole

15 104 19 98 16

3 106 20 93 14

6 114 18 107 14


6 99 15 90 12

12 120 14 91 4


3 88 13 76 8

6 90 12 89 9


6 97 12 108 14

12 101 11 117 7

Econazole 15 84 15 109 21

3 78 10 98 18

6 77 9 115 15


3 82 12 78 14

6 88 9 87 13


6 90 11 106 8

12 102 11 113 10

Fluconazole

15 91 8 92 9

3 105 5 105 9

6 106 5 96 6








Conclusions


Acknowledgments


References






Analyte












Fluconazole 5 7 5 6

















































3530 to 285144 17



3501 to 127235 14




5311 to 82450 36




2777 to 242240 16




3111 to 228048 10



7053 to 432058 32




3813 to 193335 22



4171 to 229061 24



5319 to 277139 30



3051 to 129935 11














LCMSMS Analyses





freezing filtration

Griseofulvin 77 0 0




Bifonazole 98 57 86


Econazole 95 75 83

Miconazole 94 91 56


Fluconazole 86 10 0










Itraconazole-d5 8125 15099 21745 13531 24701 12143 13917 37



Itraconazole-d5 70968 50873 10193 93354 75968 72111 33241 33


Itraconazole-d5 14575 22677 7981 14498 21126 35129 30530 46





Calculations











LCMSMS




Sample Preparation



Analyte


CCα microgkg

CCβ microgkg

CCα microgkg

CCβ microgkg





Bifonazole 005 095 014 079


Econazole 007 110 010 050

Miconazole 016 111 028 082








Method Validation



Analyte



Accuracy

Precision

Accuracy

Precision


6 107 17 110 22

12 104 16 106 19


3 93 17 105 20

6 89 15 99 8

Ketocon-azole

15 104 19 98 16

3 106 20 93 14

6 114 18 107 14


6 99 15 90 12

12 120 14 91 4


3 88 13 76 8

6 90 12 89 9


6 97 12 108 14

12 101 11 117 7

Econazole 15 84 15 109 21

3 78 10 98 18

6 77 9 115 15


3 82 12 78 14

6 88 9 87 13


6 90 11 106 8

12 102 11 113 10

Fluconazole

15 91 8 92 9

3 105 5 105 9

6 106 5 96 6








Conclusions


Acknowledgments


References






Analyte












Fluconazole 5 7 5 6













































LCMSMS Analyses





freezing filtration

Griseofulvin 77 0 0




Bifonazole 98 57 86


Econazole 95 75 83

Miconazole 94 91 56


Fluconazole 86 10 0










Itraconazole-d5 8125 15099 21745 13531 24701 12143 13917 37



Itraconazole-d5 70968 50873 10193 93354 75968 72111 33241 33


Itraconazole-d5 14575 22677 7981 14498 21126 35129 30530 46





Calculations











LCMSMS




Sample Preparation



Analyte


CCα microgkg

CCβ microgkg

CCα microgkg

CCβ microgkg





Bifonazole 005 095 014 079


Econazole 007 110 010 050

Miconazole 016 111 028 082








Method Validation



Analyte



Accuracy

Precision

Accuracy

Precision


6 107 17 110 22

12 104 16 106 19


3 93 17 105 20

6 89 15 99 8

Ketocon-azole

15 104 19 98 16

3 106 20 93 14

6 114 18 107 14


6 99 15 90 12

12 120 14 91 4


3 88 13 76 8

6 90 12 89 9


6 97 12 108 14

12 101 11 117 7

Econazole 15 84 15 109 21

3 78 10 98 18

6 77 9 115 15


3 82 12 78 14

6 88 9 87 13


6 90 11 106 8

12 102 11 113 10

Fluconazole

15 91 8 92 9

3 105 5 105 9

6 106 5 96 6








Conclusions


Acknowledgments


References






Analyte












Fluconazole 5 7 5 6





































Calculations











LCMSMS




Sample Preparation



Analyte


CCα microgkg

CCβ microgkg

CCα microgkg

CCβ microgkg





Bifonazole 005 095 014 079


Econazole 007 110 010 050

Miconazole 016 111 028 082








Method Validation



Analyte



Accuracy

Precision

Accuracy

Precision


6 107 17 110 22

12 104 16 106 19


3 93 17 105 20

6 89 15 99 8

Ketocon-azole

15 104 19 98 16

3 106 20 93 14

6 114 18 107 14


6 99 15 90 12

12 120 14 91 4


3 88 13 76 8

6 90 12 89 9


6 97 12 108 14

12 101 11 117 7

Econazole 15 84 15 109 21

3 78 10 98 18

6 77 9 115 15


3 82 12 78 14

6 88 9 87 13


6 90 11 106 8

12 102 11 113 10

Fluconazole

15 91 8 92 9

3 105 5 105 9

6 106 5 96 6








Conclusions


Acknowledgments


References






Analyte












Fluconazole 5 7 5 6








































Method Validation



Analyte



Accuracy

Precision

Accuracy

Precision


6 107 17 110 22

12 104 16 106 19


3 93 17 105 20

6 89 15 99 8

Ketocon-azole

15 104 19 98 16

3 106 20 93 14

6 114 18 107 14


6 99 15 90 12

12 120 14 91 4


3 88 13 76 8

6 90 12 89 9


6 97 12 108 14

12 101 11 117 7

Econazole 15 84 15 109 21

3 78 10 98 18

6 77 9 115 15


3 82 12 78 14

6 88 9 87 13


6 90 11 106 8

12 102 11 113 10

Fluconazole

15 91 8 92 9

3 105 5 105 9

6 106 5 96 6








Conclusions


Acknowledgments


References






Analyte












Fluconazole 5 7 5 6










































Conclusions


Acknowledgments


References






Analyte












Fluconazole 5 7 5 6



































Development of a simple liquid chromatography-tandem mass spectrometry method for multiresidue...

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