Determination of chloramphenicol in honey by liquid chromatography–tandem mass spectrometry

Analytica Chimica Acta 529 (2005) 257–263

Determination of chloramphenicol in honey by liquid chromatography–tandem mass spectrometry A.F. Forti, G. Campana, A. Simonella, M. Multari, G. Scortichini∗ Istituto Zooprofilattico Sperimentale dell’Abruzzo e del Molise “G. Caporale”, Via Campo Boario, 64100 Teramo, Italy Received 31 May 2004; received in revised form 26 October 2004; accepted 26 October 2004 Available online 29 December 2004

Abstract An effective liquid chromatographic method with tandem mass spectrometric (LC–MS/MS) detection and identification is presented for the determination of chloramphenicol (CAP) in honey. After a preliminary dissolution in water, samples were extracted with a mixture of dichloromethane/acetone and evaporated to dryness; the following clean up was carried out on an octadecyl (C18 ) SPE cartridge. CAP was determined by LC–MS/MS, using electrospray ionization in the negative ion mode (ESI− ). The column was a LUNA Phenomenex with a mixture of methanol-aqueous ammonium acetate (60:40, v/v) as a mobile phase. Honey samples were fortified at CAP levels 0.30–0.45–0.60 ␮g kg−1 with 5D-CAP as internal standard. At these levels, trueness ranged between 98.7 and 102.0% and within-laboratory reproducibility was lower than 6.2%, expressed as relative standard deviation. The limit of decision (CC␣) was 0.07 ␮g kg−1 and detection capability (CC␤) was 0.10 ␮g kg−1 . © 2004 Elsevier B.V. All rights reserved. Keywords: Chloramphenicol; Honey; Solid-phase extraction; Liquid chromatography–tandem mass spectrometry; Validation

1. Introduction Chloramphenicol (CAP) is a broad-spectrum antibiotic, exhibiting activity against a variety of aerobic and anaerobic micro-organisms. Its protein inhibiting property makes it effective in the treatment of several infectious diseases. As heavy side effects have been extensively demonstrated in humans [1] (e.g. aplastic anaemia and hypersensitivity), the European Community banned its use in food-producing animals since 1994 [2], in order to protect consumers’ health. Consequently CAP has been included in the Group A of the Council Directive 96/23/EC [3], including those substances for which a “zero tolerance residue limit” has been established in edible tissues. In spite of it, this drug is still illicitly used in animal farming because of its ready availability and low cost. Specific and sensitive analytical methods are thus required for a concrete monitoring of CAP at residual levels in animal food and other matrices including honey. For ∗

Corresponding author. Tel.: +39 0861 332242; fax: +39 0861 332251. E-mail address: [email protected] (G. Scortichini).

0003-2670/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.aca.2004.10.059

screening purposes, immunochemical tests [4–6] and chromatographic techniques such as liquid chromatography with diode array detection (HPLC-DAD) and gas chromatography with electron capture detection (GC-ECD) have been used [7–9]. However, according to the Commission Decision 2002/657/EC [10], the confirmation of suspect positive samples must be carried out by mass spectrometry coupled to the adequate chromatographic separation. This is the most reliable analytical method for the unambiguous confirmation of “zero tolerance residue limit” substances in products of animal origin. Moreover, because of its high sensitivity, the combination mass spectrometry–liquid chromatography (LC–MS) allows to meet the requirements of the recent Commission Decision 2003/181/EC, that fixed a “minimum required performance limit” (MRPL) of 0.3 ␮g kg−1 for the detection of CAP in several food items like honey [11]. Various applications of this technique for the analysis of CAP are reported in [12–16]. In the present work, a sensitive and reliable method for the determination of CAP in honey by LC–MS/MS, in compliance with the legislation in force, is described.

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2. Experimental 2.1. Chemicals 2.1.1. Reagents All reagents and solvents were of analytical or HPLC grade quality and supplied by J.T. Baker (Deventer, The Netherlands) and Carlo Erba (Milan, Italy). Ultrapure water was obtained by Milli-Q system Millipore (Bedford, MA, USA). The octadecyl (C18 ) solid-phase extraction (SPE) cartridges (500 mg/3 ml) were from J.T. Baker. The phosphate buffer solution (PBS) at pH 7.8 was prepared by mixing 8.5 ml of the solution A with 91.5 ml of the solution B. Solution A: 9.78 g of KH2 PO4 ·H2 O in 1000 ml of ultrapure water. Solution B: 11.88 g of Na2 HPO4 ·2H2 O in 1000 ml of ultrapure water. 2.1.2. Standards CAP standard was purchased from Sigma–Aldrich (St. Louis, MO, USA), while 5D-CAP (internal standard) was bought from Cambridge Isotope Laboratories (Andover, MA, USA). CAP and 5D-CAP stock solutions at the concentration of 1.0 mg ml−1 in methanol were prepared and stored at 4 ◦ C. CAP and 5D-CAP suitable working solutions used for spiking blank samples were obtained by dilution till a concentration of 0.1 ␮g ml−1 .

Fig. 1. Mass spectrum of CAP and its typical fragmentation pattern.

ing to the multiple reaction monitoring (MRM) approach, by selecting the two most intense transition reactions from CAP (321 → 152; 321 → 121) and 5D-CAP (326 → 157; 326 → 126) (Figs. 1 and 2). CAP quantitation was accomplished by the isotope dilution method considering the most intense ion transition. 2.3. Sample preparation An aliquot of 10 g of honey was spiked with 2.5 ␮g kg−1 of 5D-CAP. It was then completely dissolved in water (10 ml)

2.2. Apparatus The LC–MS/MS system consisted of a Series 200 pump and autosampler (Perkin Elmer, Norwalk, CT, USA) coupled to an API 2000 triple quadrupole mass spectrometer (Sciex, Toronto, Canada). The chromatographic separation was performed on a LUNA ODS(2) C18 3 ␮m column, 7.5 mm × 4.6 mm i.d. (Phenomenex, Torrance, CA, USA), by using an isocratic mobile phase of methanol-5 mM ammonium acetate (60:40, v/v). The flow rate was set at 0.2 ml min−1 , the injection volume at 50 ␮l and the column temperature at 40 ◦ C. The MS detector was operated in the negative ion mode with a TurboIon SprayTM source heated to 450 ◦ C. Capillary voltage was set at 4.5 kV with an orifice potential of 20 V. Nitrogen was used as a curtain and a collision gas. The collision energies were separately optimized for the two selected ion transitions of both CAP and 5D-CAP (Table 1). Data were acquired accordTable 1 Ion transitions and corresponding collision energies used for CAP quantification Analyte

Ion transitions (m/z)

Collision energy (eV)

CAP

321 → 152a 321 → 121b

22 46

5D-CAP

326 → 157a 326 → 126b

22 46

a b

More intense ion transition. Less intense ion transition.

Fig. 2. Full scan mass spectra of CAP (A) and 5D-CAP (B), also depicting their chemical structures.

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by agitation on a shaker and extracted with a mixture (50 ml) of dichloromethane–acetone (1:1, v/v) in Ultra-Turrax. A following centrifugation (5 min, 1280 g) allowed to separate the aqueous layer from the upper organic layer. Supernatant (5 ml) was evaporated to dryness under a stream of nitrogen at 50 ◦ C and the remainder was dissolved in PBS buffer (2.5 ml, pH 7.8) and loaded on the C18 SPE column, pre-activated with methanol (2.5 ml) and water (5 ml). The cartridge was washed with water (5 ml), vacuum-dried and washed with petroleum ether (5 ml). After a further evaporation under vacuum, CAP was eluted with diethyl ether (10 ml). Solvent was evaporated in nitrogen flow and the residue was dissolved in 200 ␮l of the HPLC mobile phase. The injection volume was 50 ␮l.

fold the standard deviation of the within-laboratory reproducibility at 0.3 ␮g kg−1 . Method ruggedness was estimated by means of the Youden robustness test [17]. This experimental design entails the conduction of eight experiments and the selection of seven variables, chosen in the sample preparation and analysis. The application of this test consists in the introduction of minor simultaneous changes in these parameters according to an established experimental design, with the aim of identifying the critical factors that may have to be controlled in order to obtain accurate assay results. The factors taken into account in this study and their levels of variation are reported in Table 2.

2.4. Method validation

3. Results and discussion

Method validation was carried out according to criteria set by Commission Decision 2002/657/EC. Parameters taken into account were: specificity, response linearity, trueness, precision (repeatability and within-laboratory reproducibility), decision limit (CC␣), detection capability (CC␤), ruggedness. Specificity was tested by analyzing 20 blank honey samples of different origin in order to verify the absence of potential interfering compounds at CAP retention time. Response linearity was evaluated by drawing six points calibration curves in solvent with CAP concentrations corresponding to 0, 0.625, 1.25, 2.5, 5, 10 ␮g l−1 , and containing a fixed amount of 5D-CAP (12.5 ␮g l−1 ). Each regression curve was prepared plotting the ratio CAP area (321 → 152)/5D-CAP area (326 → 157) versus CAP concentration in ␮g l−1 . Method trueness and precision were determined by spiking blank honey samples with CAP, resulting in two analytical series, each with three concentration levels (0.30–0.45–0.60 ␮g kg−1 ) and six samples per concentration level. Trueness was expressed in terms of recovery rates and precision as relative standard deviation. Decision limit (CC␣) and detection capability (CC␤) were calculated by applying the calibration curve procedure described in Commission Decision 2002/657/EC [10]. CC␣ was expressed as the concentration corresponding to the y-intercept plus 2.33-fold the within-laboratory standard deviation of the lowest calibration level (0.3 ␮g kg−1 ). CC␤ was calculated as CC␣ + 1.64-

The comparative chromatograms reported in Fig. 3 highlighted the specificity and sensitivity of the present method. CAP does not form any adduct in honey (unlike liver and kidney) and thus no preliminary deconjugation is required for the analysis. Thus, according to the described procedure, honey was dissolved in water without any enzymatic digestion or acid hydrolysis [18]. Furthermore, the choice of the appropriate clean up procedure permitted to avoid the presence of interfering peaks at CAP retention time [19], by meaning as “interfering peak” any substance naturally occurring in honey that could give arise to eventual interference in the chromatographic run. The analysis of several blank samples of different origin revealed the complete absence of these peaks and demonstrated that adopted clean up was fit for purposes. The C18 cartridge gave a good retention for CAP and the following washings were able to eliminate the presence of either polar or non-polar compounds. The adoption of the adequate internal standard was of help in compensating the ion suppression effects induced by the matrix. Ion suppression was evaluated by performing a standard addition of natural chloramphenicol to the sample just before the instrumental analysis in order to avoid the loss of analyte due to the clean up step. On the basis of this tests ion suppression can be estimated between 60 and 70%. Commission Decision 2002/657/EC set criteria for the determination of veterinary drug residues in live animals and animal

Table 2 Robustness test: selected parameters and levels Selected variable

Abbreviationa

High level

Low level

Dichloromethane concentration in the extraction mixture (%) Extract evaporation temperature (◦ C) Extract evaporation mode Buffer pH SPE cartridge elution mode SPE cartridge elution volume (ml) Ammonium acetate concentrationd (mM)

A,a B,b C,c D,d E,e F,f G,g

55 55 To dryness 8.6 Always wetb 11 5.5

45 45 To dryness + 5 min 7.0 Left to dryc 9 4.5

a b c d

Upper case letter represents high level, lower case letter represents low level value of the variable. The cartridge was kept wet at all stages during the clean up process. The cartridge was left to dry for 1 min before and after loading the sample. Aqueous component in LC–MS/MS mobile phase.

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Fig. 3. MRM chromatograms of a blank honey sample with internal standard (2.5 ␮g kg−1 ) and the same sample spiked with 0.3 ␮g kg−1 CAP and with internal standard (2.5 ␮g kg−1 ).

261

Fig. 3. (Continued).

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Table 3 Results of the regression analysis of CAP calibration curves (graphs) Curve

Slope

Standard deviation slope

Intercept

Standard deviation intercept

R2

Standard error

1 2 3

0.0228 0.0725 0.0704

0.0002 0.0008 0.0004

0.0007 0.0020 0.0041

0.0008 0.0036 0.0021

0.9998 0.9996 0.9998

0.0014 0.0064 0.0038

Table 4 Method trueness and precision Spike level (␮g kg−1 )

Repeatability (intra-day) Mean recovery (%)

R.S.D. (%)

Replicates

Mean recovery (%)

R.S.D. (%)

Replicates

0.30 0.45 0.60 Overall

98.0 98.6 99.9 98.8

5.0 4.6 2.9 4.1

6 6 6 18

102.0 98.7 100.0 100.2

6.2 4.1 2.6 4.6

12 12 12 36

Reproducibility (inter-day)

products based on a number of identification points (IPs). In this application CAP was analyzed in MRM mode by monitoring three different ions (one precursor and two fragments). This approach allowed to achieve the four IPs required by the aforesaid document for the identification of banned compounds. Regarding response linearity, the determination coefficients of the regression curves (R2 ) constructed in solvent were always >0.9996. As slopes of matrix-matched and solvent calibration curves overlapped the latter were used for quantitation purposes (Table 3). Trueness and precision data for CAP are given in Table 4. The mean recoveries (internal standard-corrected) ranged between 98.7 and 102.0%, with reproducibility relative standard deviations from 2.6 to 6.2%. In particular, precision values improved as far as the fortification levels increased. The within-laboratory reproducibility was calculated by using the single factorial analysis of variances (ANOVA). Significant differences were observed neither throughout the different spike levels for each batch of analysis nor between the two overall samples series. Calculated CC␣ was 0.07 ␮g kg−1 with a CC␤ of 0.10 ␮g kg−1 ; these values appeared very satisfying and coherent with the observation of the ion chromatograms in terms of signal-tonoise ratio for both diagnostic ion transitions as shown in Fig. 3. Seven different variables chosen within the entire analytical process were taken into account in the evaluation of method ruggedness. The experimental design was planned with the aim of identifying the critical points in the procedure. Four variables were chosen to determine the influence of minor changes in the mixture composition on the extraction efficiency and also to evaluate CAP stability in different conditions of desiccation, temperature and pH. Regarding clean up, it was verified the relationship between the CAP recovery rates and both the elution volume and the state of the cartridge (kept constantly wet or dried under vacuum for a short time). Finally it was investigated if slight variations of the salt concentration in the aqueous phase could cause significant changes in the intensity of CAP signal in the chromatograms. The effect of each factor was estimated by determining the difference between the mean result for the variable

at a “high level” (corresponding to the capital letter) and at a “low level” (indicated by the small letter) (Table 2). The standard deviation of the differences Di (SDi
) was calculated   Di2  according to the Youden approach: SDi = 2 7 . As the obtained value (4.8%) was less than the standard deviation of the within-laboratory reproducibility at 0.3 ␮g kg−1 (6.2%), it was demonstrated that all selected factors together do not significantly affect the analytical performance. Besides this overall result, the influence of each variable on method robustness was also evaluated by applying the t-test [20]. The experimental t values resulted lower than the twotailed t critical value for n − 1 degrees of freedom (tcrit = 2.20, ν = 12 − 1, 95% confidence level) for all the seven variables (Table 5). Thus, no factor had a significant effect on ruggedness. Consequently method proved to be fairly robust and able to withstand minor fluctuations in the operating variables that may occur in its routine application. The main properties of the present method are the simplicity of the extraction and clean up, thus representing an improvement on previously described methods.

Table 5 Robustness test results Variable

Difference (Di) in % Recovery absolute value

t-valuea

Dichloromethane % in the extraction mixture Extract evaporation temperature Extract evaporation mode Buffer pH SPE cartridge elution mode SPE cartridge elution volume Ammonium acetate concentration

1.60

0.36

1.90 1.45 3.85 0.00 2.20 7.25

0.43 0.33 0.88 0.00 0.50 1.65

√

t = √ n|Di| where n = 4 (number of experiment carried out at each level 2 S.D. for each parameter) and S.D. = 6.2% (standard deviation obtained from the analysis of 12 spiked samples at 0.3 ␮g kg−1 ); two-tailed critical value tcrit 2.20, ν = 12 − 1, 95% confidence level. a

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4. Conclusions The proposed method was developed for the determination of CAP in honey at the European MRPL. Tandem mass spectrometry using MRM transitions of analyte-specific fragment ions enabled a selective and sensitive detection, in agreement with the EU criteria for the confirmatory analysis of banned substances. Such method was thus validated with the aim of meeting Commission Decision 2002/657/EC requirements. Moreover, the choice of isotope-labelled CAP as an internal standard allowed an accurate and precise quantitation of the analyte, while the adoption of the adequate clean up procedure made the method suitable for routine analysis of CAP in honey. The ruggedness test, performed according to the Youden experimental design, demonstrated that the performance of the method was not influenced by minor changes in the key analytical variables. In conclusion, the proposed LC–MS/MS method is adequate for the purpose of confirmatory analysis of CAP in honey at MRPL level.

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