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Journal of Chromatography A, 1174 (2007) 63–71

Development of an improved method for trace analysis ofchloramphenicol using molecularly imprinted polymers

Brian Boyd a,∗, Henrik Bjork a, Johan Billing a, Olga Shimelis b,Sara Axelsson a, Maria Leonora a, Ecevit Yilmaz a

a MIP Technologies AB, Box 737, Lund 22007, Swedenb Supelco/Sigma Aldrich, Bellefonte, PA 16823, USA

Available online 5 September 2007

bstract

A confirmatory method is described for the determination of the illegal antibiotic chloramphenicol using a specifically developed molecularlymprinted polymer (MIP) as the sample clean-up technique. The newly developed MIP was produced using an analogue to chloramphenicol as theemplate molecule. Using an analogue of the analyte as the template avoids a major traditional drawback associated with MIPs of residual template

eeching or bleeding. The MIP described was used as a solid-phase extraction phase for the extraction of chloramphenicol from various sample

atrices including honey, urine, milk and plasma. A full analytical method with quantification by LC–MS/MS is described. The method was fullyalidated according to the European Union (EU) criteria for the analysis of veterinary drug residues.

2007 Elsevier B.V. All rights reserved.

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eywords: Chloramphenicol; Molecularly imprinted polymer; SPE; Honey; Ur

. Introduction

With growing concerns over food safety and the need toncrease sample-throughput in analytical testing laboratories,here is a constant requirement for accurate, simpler, faster andmproved analytical methods. The complexity of food matricesnd the presence of many potential interferences, require specificnd selective methods of analysis. Often LC–MS/MS is used torovide the specificity needed in order to correctly identify con-aminants in food samples. However, LC–MS/MS alone does notrovide the sensitivity and accuracy frequently required by regu-atory and food safety agencies [1]. A common technique used toomplement LC–MS/MS analysis is the pretreatment of the sam-le by clean-up methods such as solid-phase extraction (SPE),iquid–liquid extraction, supercritical fluid extraction, etc. [2].hese clean-up techniques remove many of the matrix inter-

erences allowing more sensitive and accurate analysis by an

C–MS/MS method. Solid-phase extraction is the most popularf these clean-up techniques due to factors such as convenience,ost, time saving and simplicity. A relatively new development

∗ Corresponding author. Tel.: +46 46 163904; fax: +46 46 163901.E-mail address: brian.boyd@miptechnologies.com (B. Boyd).

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021-9673/$ – see front matter © 2007 Elsevier B.V. All rights reserved.oi:10.1016/j.chroma.2007.08.072

lasma; Milk; Veterinary drug residues

n the area of SPE is the use of molecularly imprinted polymersMIPs) for the sample clean-up [3,4]. Molecularly imprintedolymers (MIPs) are engineered cross-linked polymers that canxhibit high affinity and selectivity towards a single compoundr a class of compounds. MIPs are synthesized by polymer-zation of different components. These components include aemplate (the imprint molecule), functional monomer, cross-inker, porogen and initiator. During synthesis of these imprintedolymers, specifically designed binding sites are created whichave selective affinities for the analyte of interest. MIPs are ofteneferred to as ‘artificial antibodies’ as the selectivity they exhibitor the specific analyte they are designed for often approachntibody–antigen systems [5,6]. Unlike antibodies, MIPs aretable to extremes of pH, organic solvents and temperaturehich allows for more flexibility in the analytical methods

7].The determination of chloramphenicol (CAP) is a typical

xample of a challenging analysis for drug residues in foodatrices. Chloramphenicol is an antibiotic that was originally

solated from the bacterium Streptomyces venezuelae and is

ffective against a wide range of Gram positive and Gramegative bacteria in both humans and animals [8]. Howeverotentially fatal side-effects such as aplastic anemia are known inumans [9]. The suspected carcinogenity of CAP is also thought

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o be dosage independent [10]. Due to this toxicity in humans,AP is completely banned in food producing animals within

he EU and USA [11]. A minimum required performance limitMRPL) for chloramphenicol determination was recently set byhe EU at 0.3 �g/kg in all food of animal origin [12]. Existing

ethods for the determination of CAP include immunoassays13], microbiological methods [14], sensors [15] and chromato-raphic methods using GC–MS [16] and LC–MS/MS [17].mmunoassays are often utilized as screening methods [13,18].lthough immunoassay tests are capable of indicating the pres-

nce of CAP in food samples, the high false positive and falseegative rates necessitate unequivocal confirmatory methods foruantification of the CAP [18]. Confirmatory methods for CAPre generally based on mass spectrometry detectors due to sensi-ivity required and the added advantage of analyte confirmation.rom the literature it is obvious that elaborate and tedious sam-le preparations are required for confirmatory methods [17,19].ften differences in method performance are reported due toifferent sample sources and matrix effects when LC–MS/MSetectors are used [17,20]. The presence of such matrix effectsequire tedious preparations of different sets of standards for dif-erent matrices and sample sources, for accurate quantification21].

The use of MIPs for the determination of CAP has been previ-usly described in sample matrices such as milk [22,23], serum24], honey [5] and shrimp [23]. However, these reported analyti-al methods do not have the sensitivity required to unequivocallyonfirm a positive result for CAP at or below a detection limit of.3 �g/kg. In addition, all of these publications have employedAP as the template molecule in the MIP. A well documentedrawback of MIPs is the residual template leaching or bleed-ng that may occur from the MIP even after extensive washing3,24]. This presents the problem of false positives in confir-atory analytical methods due to potential leaching of residual

emplate. To improve the reliability of using MIPs in analyti-al methods, we investigated a novel MIP which was producedsing an analogue of the analyte.

The purpose of this study was to develop a confirmatoryethod for the determination of CAP, based on the use of a MIPhich was produced using an analogue of CAP, at the detec-

ion limits required by regulatory agencies. The new methods compared to recently published methods using other samplelean-up techniques, such as liquid–liquid extraction [19] andydrophilic polymeric SPE phases [25]. Also the new MIP basedPE method is investigated with milk, urine, honey and plasmaample matrices from various sample specimens as a methodith a broad scope is preferable. The method is also validated

or honey and urine sample matrices according to EU guidelinesn the performance of confirmatory analytical methods [26].

. Experimental

.1. Chemicals

Chloramphenicol (CAP) was purchased from Sigma–AldrichSchnelldorf, Germany). Internal standard deuterated chloram-henicol, [2H5]CAP (CAP-d5) (>98% chemical purity, 99.6%

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r. A 1174 (2007) 63–71

sotopic purity) was purchased from Cambridge Isotope Labs.Andover, MA, USA). Solvents and all other chemicals were oft least HPLC-grade and were purchased from Sigma–AldrichSchnelldorf, Germany). All distilled water used was purifiedsing an ultra pure water system from Elga (High Wycombe,K). SupelMIP SPE chloramphenicol cartridges were suppliedy Sigma–Aldrich (Schnelldorf, Germany). Oasis HLB 200 mgmL cartridge (WAT 106202) was purchased from Waters (Sol-

entuna, Sweden).

.2. Chromatographic evaluation of SupelMIP SPEhloramphenicol

The SupelMIP SPE chloramphenicol was developed andynthesized at MIP Technologies, Sweden. A correspondingon-imprinted polymer (NIP) was also synthesized at MIP Tech-ologies. The non-imprinted polymer was synthesized similarlyo the SupelMIP but without the use of a template molecule.or comparison to previous MIPs synthesized for CAP, a MIPReference MIP) was also synthesized according to the proce-ure described by Levi et al. [24]. Briefly, this synthesis was asollows:

Chloramphenicol (0.32 g, 1 mmol) was dissolved inmL of tetrahydrofuran (THF). The functional monomer

diethylamino)ethyl methacrylate (DAM) (0.4 mL, 2 mmol),he cross-linking monomer ethylene glycol dimethacrylayeEGDMA) (5 mL, 26 mmol) and the initiator 2,2′-azobis(2-ethylpropionitrile) (AIBN) (0.04 g, 0.24 mmol) were added.he solution was degassed with argon for 5 min under sonica-

ion and then polymerised for 48 h in 60 ◦C. The polymer wasrushed, grinded, washed with N,N-dimethylformamide (DMF)nd boiling ethanol and sieved in ethanol. A corresponding non-mprinted polymer (NIP) was also synthesized without the usef the template molecule.

Polymer particles of the correct particle size (2–10 �m) wereacked into a HPLC column (100 mm × 4.6 mm) for the newlyeveloped SupelMIP SPE chloramphenicol, the correspondingIP and the reference MIP. A CAP solution (100 �g/mL inater) was injected onto each HPLC column. The retention ofAP on each phase was tested using a LC20 HPLC system fromhimadzu (Kyoto, Japan). The mobile phase solvent was 36%cetonitrile in 1% (v/v) ammonia (aq.) and an isocratic methodas applied with 1.0 mL/min. The injection volume was 80 �L,

he column temperature was 25 ◦C and the total run time was0 min. The UV spectrum was monitored at 215 nm.

.3. Samples

Honey samples of different geographical origin, i.e. fromSA (Florida), South America, Hungary, Argentina, Sweden

nd France, were purchased from local supermarkets or obtainedrom private beekeepers; in the later case the beehives were

Urine samples from different bovine sources were a contri-ution from SLV (Statens Livsmedelsverk), Sweden. The urineamples were centrifuged at 2000 × g for 10 min, and stored at20 ◦C in airtight containers until analysis.

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.4. Sample preparation and clean-up

.4.1. Honey samples by hydrophilic polymer SPEFor honey samples cleaned-up using the Oasis HLB

ydrophilic polymer SPE cartridges the application by Hancockas followed [25]. A 1-g amount of honey was weighed into aeaker and 1 mL of water was added to get a honey solution. Thisoney solution was placed in a water bath at 45 ◦C and heatedor 5 min. The honey sample solutions were fortified with a con-entration of 1 �g/L CAP-d5. The honey solutions were furtherxtracted with 2 mL of ethyl acetate and centrifuged for 5 mint 550 × g. The supernatant was transferred to a clean tube andvaporated at 50 ◦C to dryness. The residue was reconstituted inmL of methanol and diluted with 20 mL of water.

The samples prepared above were applied onto the SPE car-ridge using a vacuum manifold system (Supelco, Germany)t a flow rate of 1 mL/min. Before extraction, the cartridgesere conditioned with 5 mL of methanol followed by 5 mLf HPLC grade water. After loading the samples (20 mL), thePE columns were washed with 5 mL water. Slight vacuumas applied before elution of chloramphenicol was achieved,y applying 2× 2.5 mL pure methanol. A slight vacuum waspplied between the two elution aliquots. The elution aliquotsere then evaporated under vacuum at 55 ◦C for 55 min and

econstituted in 100 �L of 30% acetonitrile in 10 mM ammo-ium acetate at pH 6.7 before analysis with LC–MS/MS.

.4.2. Honey samples by SupelMIP SPE chloramphenicolA honey sample was prepared as described for the hydrophilic

olymer SPE but with the ethyl acetate extraction omitted. Theamples were applied onto the SupelMIP SPE chlorampheni-ol cartridge using a vacuum manifold system at a flow rate of.5 mL/min. Before extraction, the SupelMIP cartridges wereonditioned with 1 mL of methanol followed by 1 mL of HPLCrade water. After loading the samples, the SPE cartridgesere washed with the following successive wash solutions:× 1 mL water, 1 mL 5% acetonitrile/95% acetic acid (0.5%, v/v,q.), 2× 1 mL 1% (v/v) ammonia (aq.) and 1 mL 20% acetoni-rile/80% ammonia (1%, v/v, aq.). The SupelMIP was then driedy applying vacuum for 5 min and another wash of 2× 1 mL% (v/v) acetic acid in dichloromethane was applied beforeurther drying for 2 min under vacuum. Elution of chloram-henicol was achieved by applying 2× 1 mL 10% (v/v) methanoln dichloromethane. The elution aliquots were then evaporatednder vacuum at 35 ◦C for 35 min and reconstituted in 100 �L of0% acetonitrile in 10 mM ammonium acetate at pH 6.7 beforenalysis with LC–MS/MS.

.4.3. Honey samples by liquid–liquid extraction (LLE)For clean-up using liquid–liquid extraction, the procedure

y Thorsen Ronning et al. [19] was followed. A 1-g amount ofoney was weighed into a beaker and 1 mL of water was added toet a honey solution. This honey solution was placed in a water

ath at 45 ◦C and heated for 5 min. The honey sample solutionsere fortified with a concentration of 1 �g/L CAP-d5. To theissolved honey 5 mL acetonitrile was added, the sample wasortexed and centrifuged at 400 × g for 5 min. The supernatant

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. A 1174 (2007) 63–71 65

as mixed with 5 mL of chloroform, vortexed and centrifugedor 3 min at 250 × g. The organic layer was evaporated, and theample reconstituted into 100 �L of 30% acetonitrile in 10 mMmmonium acetate at pH 6.7 before analysis with LC–MS/MS.

.4.4. Urine samples by SupelMIP SPE chloramphenicolFor urine samples, the pH of the samples was adjusted with

cetic acid to a final pH between 7.0 and 7.5. The samplesere then fortified with 1 �g/L CAP-d5. 1 mL of each urine

ample was then cleaned up as described for the honey sam-les by SupelMIP SPE chloramphenicol. However elution ofhloramphenicol for urine samples was achieved by applying× 1 mL methanol. The elution aliquots were then evaporatednder vacuum at 55 ◦C for 35 min and reconstituted in 100 �L of0% acetonitrile in 10 mM ammonium acetate at pH 6.7 beforenalysis with LC–MS/MS.

.4.5. Milk and plasma samples by SupelMIP SPEhloramphenicol

Raw milk samples (5 mL) were centrifuged at 1100 × g for5 min. The supernatant was collected for application to theupelMIP SPE chloramphenicol cartridge. For plasma samplesnd semi-skimmed milk, no pre-treatment was required. Theamples were fortified with 1 �g/L CAP-d5. For clean-up, 1-L samples were treated as described for the honey samples byupelMIP SPE chloramphenicol with the exception that elutionf chloramphenicol was achieved by applying 2× 1 mL 89%v/v) methanol/1% (v/v) acetic acid/10% (v/v) water. The elu-ion aliquots were then evaporated under vacuum at 55 ◦C for5 min and reconstituted in 100 �L of 30% acetonitrile in 10 mMmmonium acetate at pH 6.7 before analysis with LC–MS/MS.

.5. HPLC–MS/MS analysis

Measurements were performed with a HPLC–MS/MS sys-em. HPLC analyses were performed on a Supelco Ascentis18 column (100 mm × 2.1 mm, 3 �m particle size) supplied byigma–Aldrich (Schnelldorf, Germany), using a LC20 HPLCystem from Shimadzu. The mobile phase solvent was 30%v/v) acetonitrile in 10 mM ammonium acetate, pH 6.7 and ansocratic method was applied with 0.2 mL/min. The injectionolume was 20 �L, the column temperature was 25 ◦C and theotal run time was 5 min.

For the MS/MS detection an API 3200 triple quadrupoleass spectrometer (Applied Biosystems/MDS SCIEX, Ontario,anada) equipped with Turbo V source housing was used. Nitro-en was used for the nebulizer and collision gases at pressures of0 and 30 psi, respectively. The TurboIonSpray and curtain gasesad settings of 4 and 10 psi. The source temperature used was00 ◦C, and the electrospray voltage was −2000 V. The declus-er potential was set at −35 V. Each transition had a dwell time of50 ms. The collision energy was 28 V and the entrance poten-ial was 10 V. CAP was detected by electrospray in the negative

ultiple reaction monitoring modes (MRM) by the Sciex Ana-yst software. The CAP transitions used for quantification wereased on selecting the precursor ion and monitoring two productons. The transitions monitored for CAP were: m/z 321 → 152

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for quantitation) and m/z 321 → 257 (for identification). Forhe internal standard CAP-d5 (I.S.) m/z 326 → 157 was mon-tored. Furthermore as chloramphenicol contains two chlorinetoms, additional transition reactions m/z 323 → 152 and m/z23 → 257 for CAP and 328 → 157 for CAP-d5 were recorded.

.6. Validation

.6.1. Calibration and linearityCalibration curves based on response (response ratio versus

mount ratio) were constructed from five calibration levels. Sam-les were prepared by spiking blank matrix samples of honey,rine, milk and plasma with CAP corresponding to concentra-ions of 0, 0.05, 0.1, 0.3 and 1.0 �g/kg. Internal standard withconcentration of 1 �g/L CAP-d5 was added to each sample.eak area ratios of [CAP]/[d5-CAP] were plotted against thedded CAP concentrations ([CAP]) and the intercept, the stan-ard deviation and RSD were then calculated. Quantification ofAP in honey and urine samples was done using the calibra-

ion curves from m/z 321 → 152. The m/z 321 → 257 transitionas used for confirmation of results from the m/z 321 → 152

ransition.

.6.2. Repeatability/intermediate reproducibilitySix blank urine and honey samples spiked with CAP at three

evels (0.05, 0.1 and 0.3 �g/kg) were prepared. Internal standardith a concentration of 1 �g/L CAP-d5 was added to each sam-le. The samples were treated as described in Section 2.3 andhe recovery of CAP was calculated using the calibration curves.

To determine intermediate reproducibility (within-aboratory), six blank urine and honey samples spikedith CAP at three levels (0.05, 0.1 and 0.3 �g/kg) wererepared by a different analyst on a different day. The samplesere treated as described in Section 2.3 and the extracts were

nalysed using a different LC–MS/MS system. Recovery ofAP was calculated using the calibration curves.

.6.3. Detection limitsSix blank honey samples and six blank urine samples were

repared and treated as described in Section 2.3. The CCα andCβ were calculated from six blank honey samples and six blankrine samples quantified against the calibration curve from theinearity testing.

.6.4. AccuracyStatistical calculations for both the SupelMIP and hydrophilic

olymer SPE extraction methods for honey and urine were testedo reveal the accuracy of each method (one-side test). Six honeynd six urine samples were spiked with 0.3 �g/kg CAP. Internaltandard with a concentration of 1 �g/L CAP-d5 was added toach sample. All samples were extracted with both SupelMIPnd hydrophilic polymer SPE columns as described in Section

.3. A further test of the accuracy of the methods was also eval-ated by testing an incurred honey sample. The incurred honeyample was similarly treated and the concentration calculatedrom the calibration curve.

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r. A 1174 (2007) 63–71

.6.5. Matrix effectsTo investigate the effects due to sample matrix on CAP,

ve blank samples of honey were extracted according to theupelMIP and hydrophilic polymer protocols outlined in Sec-

ion 2.3. Prior to evaporation the extracts were spiked with CAPt five different concentrations (0, 0.1, 1, 2, and 5 �g/kg). Inter-al standard with a concentration of 1 �g/L CAP-d5 was alsodded to each sample. A corresponding calibration curve waschieved by spiking pure elution solvent with the same amountsf CAP (0, 0.1, 1, 2, and 5 �g/kg) and internal standard, 1 �g/LAP-d5. Four blank honey samples were extracted according

o the liquid–liquid extraction (LLE) protocol outlined in Sec-ion 2.3. Prior to evaporation the extracts were spiked with CAPt four different concentrations (0, 1, 2, and 5 �g/kg). Internaltandard with a concentration of 1 �g/L CAP-d5 was also addedo each sample. All samples were then evaporated under vac-um at 55 ◦C for 35 min and reconstituted in 100 �L of 30%cetonitrile in 10 mM ammonium acetate at pH 6.7 before anal-sis with LC–MS/MS. CAP peak areas from extracted samplesnd elution solvent spiked with CAP were plotted against CAPoncentration.

.6.6. Scope and applicationTo determine the applicability of the SupelMIP SPE chloram-

henicol method, different sample matrices and samples fromifferent sources, were investigated. Seven honey samples fromSA (Florida), South America, Hungary, Argentina, Sweden,on-EU (country not specified) and France were collected. Fiveovine urine samples from Specimens A–E were collected. Twouman plasma samples from individuals A and B were collected.wo human urine samples from individuals A and B were col-

ected. Three different milk samples (raw milk, semi-skimmedilk and UHT milk) were also collected. All samples were ini-

ially tested as blanks to confirm that no chloramphenicol wasresent in the samples. All samples were spiked with CAP at aevel of 1 �g/kg. Internal standard with a concentration of 1 �g/LAP-d5 was added to each sample. All samples were treated asescribed in Section 2.3 and the recovery of CAP was calculatedsing the calibration curves.

. Results and discussion

.1. Development of the SupelMIP SPE forhloramphenicol

All of the previously reported publications involving CAPIPs [5,22–24] have employed CAP as the template molecule.

o reduce the risk of residual template leaching or bleedinghat may occur from the MIP extensive washing is performed24]. While the advances in washing techniques have dramati-ally improved, low template bleeding levels still remain (in theg/kg range) [22–24]. The washing alternative is not an option

or an analytical SPE MIP that requires detection limits in the

g/kg range or better due to the risk of potential false posi-

ives even from low levels of template bleeding. For this reason,he SupelMIP chloramphenicol investigated here was producedsing an analogue of the analyte.

atogr. A 1174 (2007) 63–71 67

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To establish the selectivity of a MIP for a particular analyte,non-imprinted polymer (NIP) is often synthesized in paral-

el with the MIP synthesis [3]. A non-imprinted polymer (NIP)onsists of the same constituents as the MIP except the tem-late molecule is not included in the production. As the templateolecule is responsible for the formation of the specific binding

ites in the MIP, then a comparison of the MIP and NIP will revealhe extent of selective binding on the MIP for the analyte. Thisifference is often referred to as imprinting strength or imprint-ng factor [3,5]. Levi et al. [24] have previously reported the goodxtraction performance of a CAP MIP produced with CAP asemplate. They have also shown the good selectivity of the MIPompared to a corresponding NIP. A CAP MIP was synthesizedccording to Levi’s method [24], as a Reference MIP for com-arison to the SupelMIP SPE chloramphenicol described here,hich was synthesized with an analogue of chloramphenicol.With the HPLC conditions described in Section 2, an

mprinting factor of 2.43 was measured for the referencehloramphenicol MIP relative to the corresponding NIP. Thisignifies a strong selective binding in the imprinted sites of the

IP for CAP. An imprinting factor of 1.93 was measured for theupelMIP SPE chloramphenicol, manufactured with an analytenalogue of CAP, when compared with its corresponding NIP.s expected these Imprinting Factors indicate that retention ofAP on the reference MIP is stronger than on the SupelMIP SPEhloramphenicol. Imprinted sites created using chloramphenicols the template will lead to stronger rebinding of chloram-henicol than on a MIP produced with a non-chloramphenicolemplate. However the imprinting factor of the new MIP towardsAP is still significant. This indicates the new MIP made withn analogue of CAP as the template, also has selective strongnteractions similar to a MIP made with CAP as the template.his is a significant development in the use of analogues of thenalyte as templates in MIPs. The synthesis of the SupelMIPPE chloramphenicol with an analogue of CAP as the template

s a breakthrough development for the use of the MIP in annalytical SPE confirmatory method for CAP.

.2. Sample clean-up and extraction

Extraction methods used for honey and urine were comparedetween the new SupelMIP SPE chloramphenicol cartridges andecently published methods which use a LLE method [19] andtandard hydrophilic polymer SPE phase [25].

In order to compare the cleanliness of elutes from honeyxtracts for the different clean-up methods, a total ion scan waserformed. Fig. 1a and b shows comparisons of total ion scans ofoney extracts which have been cleaned-up with an hydrophilicolymer SPE cartridge, SupelMIP SPE chloramphenicol car-ridges and LLE. Superior sample cleanup was achieved withupelMIP SPE chloramphenicol cartridges compared to both

he LLE and hydrophilic polymer SPE cartridges. This is evi-ent from the number of interference mass ions in the scan of the

xtracts from the LLE and hydrophilic polymer SPE clean-ups.

The improved cleanliness of extracts from the SupelMIP SPEhloramphenicol can be explained due to the selective wash-ng solvents used in the SPE sample clean-up. The critical step

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00–400 amu. (b) Comparison of honey extracts from SupelMIP SPE chloram-henicol and a LLE sample clean-up. A total ion scan was performed over50–500 amu.

n any MIP-based SPE protocol is the selection of appropriateashing solvents, since they allow the high selectivity of the

mprinted sites to be revealed. In the SupelMIP SPE chloram-henicol method, 2 mL of ultra pure water was used for washingfter extraction to take away salts and sugars. Five percent ace-onitrile/95% acetic acid (0.5%, v/v, aq.) elutes basic interferingontaminants and 1% ammonia (aq.) takes acidic interferingontaminants away from the column. Twenty percent acetoni-rile/80% ammonia (1%, v/v, aq.) washes out hydrophobic acidicompounds while 2% (v/v) acetic acid in dichloromethane takesway the non-polar interferences. In addition, the elution sol-ent of 10% methanol in dichloromethane was used for honey.ethanol was chosen as an elution solvent for urine samples

s cleaner extracts from urine were observed using methanol ashe solvent. These wash solutions lead to the cleaner extractsbserved for the SupelMIP chloramphenicol compared to bothhe LLE and hydrophilic polymer SPE clean-up methods.

.3. Matrix effects

Many investigations into analytical troubleshooting encoun-ered with LC–MS/MS detection have focused on the problems

hich arise due to matrix effects, and in particular ion sup-ression [27]. The phenomenon of ion suppression results ineduction of signal intensity and consequently inferior per-ormance of the analytical method with regard to sensitivity,

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recision and accuracy. The cause of ion suppression is a changen the spray droplet solution properties in the MS ion source.his is due to co-extracted non-volatile or less volatile inter-

erences. To establish if matrix effects were present in thextracts, comparison tests between the LLE, SupelMIP andydrophilic polymer sample clean-up methods were performed.he ion suppression effects of all methods were evaluatedy a typical experimental system [27]. Blank samples werextracted using the appropriate procedure and spiked with CAPirectly before analysis. These spiked ion suppression sam-les, which are often referred to as matrix-matched samples,re then compared to standards prepared in solvent. The curvesor a representative honey sample extracted using the SupelMIPPE chloramphenicol, liquid–liquid extraction and hydrophilicolymer protocols are shown in Fig. 2. The actual experimen-al concentrations obtained from the calibration curves for the

atrix-matched samples are compared to the theoretical valuesor the spiked solvent standards. The experimental concentra-ions for the hydrophilic polymer and LLE clean-up proceduresre significantly lower than the theoretical values. This is dueo suppression of the CAP signal from co-extracted interfer-nces when analysed with the LC–MS/MS method. This mayead to the true level of CAP in the sample being underesti-

ated or low concentrations of CAP not being detected. Theres good agreement between the theoretical and experimental val-es for the honey extracts cleaned-up using the SupelMIP SPEhloramphenicol.

.4. Validation

.4.1. IdentificationThe SupelMIP SPE chloramphenicol SPE method for quanti-

ation of CAP was validated in honey and urine sample matrices

ig. 2. Comparison of ion suppression for SupelMIP SPE chloramphenicol,ydrophilic polymer SPE and LLE sample clean-up methods. Experimentalalues and theoretical values are plotted based on calibration curves of the matrixatched extracted honey samples and elution solvent samples, spiked with CAP.

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r. A 1174 (2007) 63–71

n order to establish the method performance. Firstly, to con-ider a positive detection of CAP in honey or urine, the criteria asescribed by Mottier et al. [17] were utilized. The criteria are (1)he retention time of the analyte varied within 1% of the retentionime of the deuterated internal standard (time: 4.0 min), (2) theresence of a signal at each of the MRM transition reactions forAP and CAP-d5, (3) 37Cl/35Cl ratios for m/z 323 → 152 versus/z 321 → 152 and m/z 323 → 257 versus m/z 321 → 257, wereithin 0.650 ± .15 and 0.330 ± .2, respectively. These qualify-

ng criteria comply with the EU guidelines for confirmatorynalytical methods [19]. All samples in this study were anal-sed according to these criteria and only when all three criteriaere confirmed was the result valid for CAP.

.4.2. Calibration and linearityThe acceptability of linearity data defined by the EU is that

correlation coefficient of >0.9980 is required for confirmatoryethods [26]. Acceptable linearity was obtained for calibration

urves in matrix-matched samples with a slope and a correlationoefficient for m/z 321 → 152 of 0.6733 and 0.9995 for honeynd 0.6954 and 0.9998 for urine, respectively. Similar slopesnd correlation coefficient were obtained for m/z 321 → 257f 0.6870 and 1.0001 for honey and 0.6900 and 0.9997 forrine. Quantification of CAP in honey and urine samples wasone using the calibration curves from m/z 321 → 152. The m/z21 → 257 transition was used for confirmation of results fromhe m/z 321 → 152 transition.

.4.3. Repeatability/intermediate reproducibilityRepeatability and intermediate reproducibility (within-

aboratory) was calculated for the SupelMIP method from thenalysis of six blank urine and honey samples spiked with CAPt three levels (0.05, 0.1 and 0.3 �g/kg). The extraction recoveryor CAP and precision (within day) for spiked honey and urineamples are summarized in Table 1. Results show that the tworansition reactions provide comparable results. At a fortifica-ion level of 0.05 �g/kg the precision values were calculated atelow 6.0% for both urine and honey. In general the recoveriesrom the honey samples are slightly higher than the urine sam-les. On further investigation this was found to be due to higheron content in the urine samples which affects the retention ofhe CAP on the SupelMIP SPE chloramphenicol.

.4.4. Detection limitsThe sensitivity of an analytical method is generally estab-

ished from the limit of detection (LOD) and the limit ofuantification (LOQ) of the method. However as there is noaximum recommended limit (MRL) for CAP [26], it is more

elevant to report the sensitivity of the method as CCα and CCβ.Cα is a measure of the lowest concentration level that can beetermined within an error probability of α. Since CAP is listedn Group A of Annex 1 of Council 96/23/EC31 [17], α = 1% forAP. CCβ is the minimum concentration of the substance that

an be detected, identified and quantified in a sample with anrror probability of β [17], where β = 5% for CAP. Accordingo Annex 1 of Council 96/23/EC31, the corresponding concen-ration at y-intercept plus 2.33 times the standard deviation of

B. Boyd et al. / J. Chromatogr. A 1174 (2007) 63–71 69

Table 1Repeatability and intermediate reproducibility of recovery rates for honey and urine samples, spiked at three different levels (six independent replicates for eachlevel)

Spike levels (�g/kg)

0.05 0.05 0.10 0.10 0.30 0.30

HoneyTransition reactions (m/z) 321 → 152 321 → 257 321 → 152 321 → 257 321 → 152 321 → 257

RepeatabilityRecovery (%) ± SD (n = 6) 81.3 ± 3.4 82.3 ± 3.9 74.6 ± 6.4 76.0 ± 3.2 75.0 ± 7.4 72.9 ± 6.9Precision 4.2 4.8 8.6 4.2 9.9 9.4

Intermediate reproducibilityRecovery (%) ± SD (n = 6) 86.2 ± 4.5 85.1 ± 4.9 78.9 ± 5.2 77.6 ± 4.8 76.5 ± 6.6 77.0 ± 9.6Intermediate reproducibility 4.1 5.4 4.0 2.4 1.4 3.9

UrineTransition reactions (m/z) 321 → 152 321 → 257 321 → 152 321 → 257 321 → 152 321 → 257

RepeatabilityRecovery (%) ± SD (n = 6) 69.9 ± 2.7 76.4 ± 3.6 68.8 ± 4.2 67.0 ± 0.5 69.1 ± 1.1 72.5 ± 2.4Precision 3.8 4.6 6.1 0.8 1.6 3.3

Intermediate reproducibility

t(ittarfl0

3

p

Fsc2

h(Chfbp0aboth sample matrices are outlined in Table 2. The SupelMIP SPE

Recovery (%) ± SD (n = 6) 73.0 ± 7.2 72.1 ± 6.9Intermediate reproducibility 3.8 4.4

he reproducibility of the intercept (s) equals the decision limitCCα = y-intercept + 2.33s) [17]. Whereas the detection capac-ty (CCβ) is the corresponding concentration at CCα plus 1.64imes the relative standard deviation of the reproducibility ofhe intercept (CCβ = y-intercept + (2.331 + .64)s) [17]. The CCα

nd CCβ for both honey and urine were 0.02 and 0.03 �g/kg,espectively. A detection limit of below 0.3 �g/kg and 0.3 �g/Lor CAP in honey and urine matrices is required due to guide-ines set by the EU [12]. An extracted honey sample spiked with.05 �g/kg is depicted in Fig. 3.

.4.5. AccuracyStatistical calculations for both the SupelMIP SPE chloram-

henicol and hydrophilic polymer SPE extraction methods for

ig. 3. The chromatogram of the 321/152 peak for an extracted honey samplepiked with 0.05 �g/kg treated with SupelMIP SPE chloramphenicol samplelean-up. The sample was analysed according to the method detailed in Section.

ct

TC(r

H

U

Am

70.6 ± 6.8 71.2 ± 4.2 77.2 ± 5.6 58.2 ± 8.42.7 6.4 11.0 15.5

oney and urine were tested to reveal the accuracy of each testone-side test). Six honey samples were spiked with 0.3 �g/kgAP and extracted with both SupelMIP chloramphenicol andydrophilic polymer SPE columns as described in Section 2. Aurther test of the accuracy of the methods was also evaluatedy testing an incurred honey sample. The incurred honey sam-le had been previously determined to have a concentration of.4 �g/kg CAP by an independent analytical laboratory usingnother analytical method. The results of the accuracy test for

hloramphenicol method provides better accuracy and precisionhan the hydrophilic polymer method. While the hydrophilic

able 2omparison of recovery rates for honey and urine samples, spiked at 0.3 �g/kg

six independent replicates for each level) extracted on SupelMIP SPE Chlo-amphenicol and hydrophilic polymer SPE columns

SupelMIP Hydrophilic polymer

oneyTransition reactions (m/z) 321 → 152 321 → 152

RepeatabilityRecovery (%) ± SD (n = 6) 69.1 ± 1.1 63.8 ± 5.8Precision 1.6 9.1

rineTransition reactions (m/z) 321 → 152 321 → 152

RepeatabilityRecovery (%) ± SD (n = 6) 72.8 ± 2.3 33.3 ± 8.7Precision 3.2 26.8

Incurred honey sample (0.4 �g/kg)Result 0.38 ± 0.04 0.21 ± 0.13Accuracy 95.0 52.5

n incurred honey sample (0.4 �g/kg) was also tested by both SPE clean-upethods for comparison to an independent analysis.

70 B. Boyd et al. / J. Chromatog

Table 3Results from analysis of different sample matrices and samples from differentspecimens using sample clean-up with SupelMIP SPE chloramphenicol

Sample matrix Source Result (spiked 1.0 �g/kg) ± SD (n = 6)

Honey USA 1.14 ± 0.01Honey Hungary 1.09 ± 0.02Honey France 1.04 ± 0.01Honey Non-EU 1.20 ± 0.01Honey South America 1.13 ± 0.03Honey Sweden 1.08 ± 0.03Honey Argentina 1.00 ± 0.02Urine Bovine A 0.91 ± 0.02Urine Bovine B 1.04 ± 0.04Urine Bovine C 1.11 ± 0.04Urine Bovine D 0.57 ± 0.06Urine Bovine E 1.06 ± 0.05Urine Human A 0.94 ± 0.02Urine Human B 0.98 ± 0.02Milk Raw milk 0.89 ± 0.03Milk Semi-skimmed 0.94 ± 0.04Milk UHT milk 0.98 ± 0.02Plasma Human A 0.93 ± 0.04Plasma Human B 0.94 ± 0.03

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hree replicates for each sample were spiked at 1 �g/kg CAP. Results areeported for the 321 → 152 transition of the LC–MS/MS method described inection 2.

olymer method is comparable in terms of repeatability foroney samples, the SupelMIP SPE chloramphenicol is also ableo accurately quantify CAP in urine samples. The experimentalesult obtained using the SupelMIP SPE chloramphenicol for thencurred honey sample (0.38 �g/kg) is in close agreement to thexpected result (0.4 �g/kg) obtained from another independentaboratory using a separate validated analytical method.

.4.6. Scope and applicationTo determine the applicability of the SupelMIP SPE chlo-

amphenicol to samples from different sources and samples withifferent matrices, a number of different samples were spikedith CAP and cleaned-up using the SupelMIP SPE chloram-henicol followed by analysis with LC–MS/MS. The results forhe different samples are summarized in Table 3. Apart fromovine sample D which was very viscous and difficult to perco-ate through the column, the results are in good agreement withhe expected spiked level. These results confirm the ability of theupelMIP SPE chloramphenicol to extract CAP from samplesrom various specimens and different sample matrices withoutloss of method performance.

. Conclusions

A fast, accurate and selective analytical method has beeneveloped for the confirmation of chloramphenicol. The methodmploys a molecularly imprinted polymer designed for extrac-ion of chloramphenicol as an SPE clean-up step prior to analysis

y LC–MS/MS. An analogue of CAP was used as the templaten the production of the MIP in order to avoid a major tradi-ional problem associated with MIPs of template bleeding. Theelectivity of the MIP allows for cleaner extracts to be obtained

[

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r. A 1174 (2007) 63–71

hich permits better detection limits and dramatically reducedffects due to the sample matrix. The method was shown to yieldore accurate and more sensitive data than methods using con-

entional hydrophilic polymeric SPE phases and liquid–liquidxtractions published previously. The method was also showno be applicable to various sample matrices and different samplepecimens. The method was validated for honey and urine sam-le matrices according to EU criteria for confirmatory analyticalethods.

cknowledgements

We thank Anna Ryberg and Eva Rustander for developmentf the MIP material.

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