Development and validation of a method for determination of corticosteroids in pig fat using liquid chromatography–tandem mass spectrometry

Journal of Chromatography B, 879 (2011) 403–410

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Journal of Chromatography B journal homepage: www.elsevier.com/locate/chromb

Development and validation of a method for determination of corticosteroids in pig fat using liquid chromatography–tandem mass spectrometry Ádám Tölgyesi a,c,∗ , Virender K. Sharma b , Jeno˝ Fekete c a b c

Central Agricultural Office Food and Feed Safety Directorate, Food Toxicology National Reference Laboratory, Budapest, Mester utca 81, 1095, Hungary Chemistry Department, Florida Institute of Technology, 150 West University Boulevard, Melbourne, FL 32901, USA Budapest University of Technology and Economics, Department of Inorganic and Analytical Chemistry, Budapest, Szt. Gellért ter 4, 1111, Hungary

a r t i c l e

i n f o

Article history: Received 24 August 2010 Accepted 23 December 2010 Available online 31 December 2010 Keywords: Corticosteroid Optimization Liquid chromatography–tandem mass spectrometry Fat Validation

a b s t r a c t A new method was developed to determine five corticosteroids (prednisolone, methylprednisone, flumethasone, dexamethasone, and methylprednisolone) in pig fat samples by liquid chromatography–tandem mass spectrometry (LC–MS/MS) utilizing an optimized liquid–liquid extraction (LLE) and subsequent solid-phase extraction (SPE) for sample clean-up. In the sample preparation, a pig fat sample was dissolved in n-hexane and then extracted into the methanol–water (50/50, v/v) mixture that enabled extraction of only medium polar corticosteroids and not the non-polar components of matrices. This extract was cleaned-up and concentrated on polymeric Oasis HLB SPE cartridge. Separation involved isocratic solvent (methanol–acetate buffer, pH 5.4) and Ascentis Express Fused-Core type HLPC column; reduced the analysis time to 7.5 min, which is at least two times lower than time required for separation using conventional techniques. Other advantage of the developed method is the minimized ion suppression of LC–MS/MS analysis, which allowed detection of corticosteroids in sub ␮g/kg. Method was validated according to European Union (EU) Commission Decision 2002/657/EC. Measured parameters such as selectivity, linearity, recovery, within-laboratory reproducibility, decision limit, and detection capability satisfied the EU Directive. Ranges of mean recoveries and within-laboratory reproducibility were 81–100% and 8.0–20.5%, respectively. Decision limits were calculated in the range from 4.5 to 11.9 ␮g/kg for MRL compounds and varied from 0.1 to 0.2 ␮g/kg for banned substances. Limit of detections (LODs), calculated as three time signal-to-noise ratio, were in the range of 0.1–0.3 ␮g/kg. © 2010 Elsevier B.V. All rights reserved.

1. Introduction Corticosteroids are widely used in veterinary medicine as anti-inflammatory. The most applied corticosteroids include prednisolone (PRED), methylprednisolone (METPRED), flumethasone (FLU), and dexamethasone (DXM) (Table 1). Medium polar corticosteroids can possibly accumulate in non-polar fatty tissues or in fat of the body, therefore, the European Union (EU) has established maximum residue limit (MRL) for permitted levels of corticosteroids in different matrices (Table 1) [1]. The values given in Table 1 are for bovine species, but the focus of the present paper is on the pig fat due to its high human consumption compared to bovine fat. Cases in which the EU has not established either MRL or MRPL (minimum required performance limit) for a compound yet, the National Reference Laboratories (NRLs) in the EU has established individually limit, which is called minimum required performance level (mrpl).

∗ Corresponding author at: Central Agricultural Office Food and Feed Safety Directorate, Food Toxicology National Reference Laboratory, Budapest, Mester utca 81, 1095, Hungary. Tel.: +36 30 9689346. E-mail address: [email protected] (Á. Tölgyesi). 1570-0232/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.jchromb.2010.12.027

The detection capability of the screening method is equal to mrpl, which is not equal to MRPL [2]. In Table 1, MRL is also given for methylprednisone (METPREDON), which is an ineffective metabolite of METPRED [3]. However, no value of METPREDON has been established until now, we applied an MRL for METPREDON as well. Because the EU might establish a sum-MRL in the future that also includes METPRED and METPREDON. This paper has used 10 ␮g/kg MRL value for METPREDON and mrpls of 1 ␮g/kg for FLU and DXM for validation of the developed method (Table 1). Recently, we have demonstrated the use of the liquid chromatography–tandem mass spectrometry (LC–MS/MS) technique to determine corticosteroids, androgens, and progesterone in biological and environmental samples [4–6]. Innovative approaches of solid phase extraction for sample preparations were performed to quantify compounds in a sub ␮g/L limit of detection (LOD). To the best of our knowledge, no LC–MS/MS method for analysis of corticosteroids in pig fat samples has been known in the literature. The objective of this paper is thus to develop method for determining corticosteroids in pig fats using LC–MS/MS technique. Pig fat contains fatty acids such as oleic, palmitic, linoleic and stearic [7], which create lipophilic environment in the sample.

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Table 1

Structures and log P values of corticosteroids. Veterinary drugs, which are corticosteroidal active ingredient content. MRLs in EU, chosen MRL for METPREDON and mrpl of no MRL substances in pig fat. MRLs, which are established for bovine fat by EU, were also applied for pig fat. Corticosteroids

Abbreviation

Prednisolone

PRED

C6

C9

C11

C16

OH

Methylprednisone

METPREDON

CH3

Flumethasone

FLU

F

O F

OH

CH3

log P

Medicinal

Animal species

Target tissues

1.64

Temaril-P® , Delta Albaplex® , PrednisTab®

bovine

milk muscle, fat liver, kidney

6 4 10

1.24

–

pig

fat

10

1.42

Flucort® , Anaprime®

pig

fat

–

milk muscle, kidney liver fat

0.3 0.75 2 –

fat, muscle, liver, kidney

1.83

Azium , Naquasone® , DexiumTM , Zonometh

bovine bovine, porcine, equidae pig

1.83

Medrol® , Cortaba®

bovine

®

Dexamethasone

DXM

Methylprednisolone

METPRED

F

CH3

OH

OH

CH3

The important step is the extraction from and clean-up of the fat matrix before analyzing corticosteroids. The known procedures for a sample preparation [4–6,8–15] are not suitable for corticosteroids analysis to sub ␮g/kg levels in pig fat samples because of the basic need of a relatively matrix free sample to avoid the ion suppression or ion enhancement in the LC–MS/MS analysis. In the present study, a simple and fast method was developed for the quantitative analysis of corticosteroids in pig fats. Liquid–liquid extraction (LLE) and solid-phase extraction (SPE) methods were tested in the sample preparation. Additionally, a shell-type fast analytical column (Ascentis Express) was applied to improve the selectivity between fatty acid matrices and corticosteroids and to minimize the matrix effect of fatty acids in multimode ion source (MMI) in the MS/MS analysis, which allowed lowering LODs to less than 1 ␮g/kg of corticosteroids. Finally, the method was validated according to the EU Commission Decision 2002/657/EC [16]. The measured analytical parameters were selectivity, ion ratios, linearity, recovery, within-laboratory reproducibility, decision limit (CC␣) and detection capability (CC␤), which met the EU directive. 2. Experimental 2.1. Reagents and samples A 1 mg/mL stock solution of methylprednisone (17,21dihydroxy-6␣-methylpregna-1,4-diene-3,11,20-trione) in ethanol was obtained from CRL RIVM (Bilthoven, The Netherlands). Other corticosteroids were prednisolone (11␤,17,21-trihydroxypregna-1,4-diene-3,20-dione], flumethasone (6␣,9␣-difluoro-11␤,17,21-trihydroxy-16␣-

MRLs (␮g/kg)

10

mrpl (␮g/kg) –

1 – 1 –

methylpregna-1,4-diene-3,20-dione), dexamethasone (9␣-fluoro-11␤,17,21-trihydroxy-16␣-methylpregna1,4-diene-3,20-dione], and methylprednisolone (11␤,17,21-trihydroxy-6␣-methylpregna-1,4-diene-3,20-dione), which were purchased from Sigma–Aldrich (Budapest, Hungary). HPLC grade methanol, ethanol, acetonitrile, dichloromethane, nhexane, ethyl acetate were obtained from Promochem (Budapest, Hungary). Methanol and acetic acid for LC–MS/MS analysis were ultrapure and were purchased from Merck (Budapest, Hungary). Ammonium acetate (99.999%) was obtained from Sigma–Aldrich (Budapest, Hungary). Stock solutions were prepared by dissolving 25 mg standards (of accurate weight) in 25 mL of methanol to obtain concentrations of 1 mg/mL and were stored at −20 ◦ C. For the working standard solutions, 25 ␮L of the stock solutions were diluted with methanol to 25 mL in volumetric flasks to yield a final concentration of 1 ␮g/mL. Working standard solutions were prepared weekly and were stored at 4 ◦ C. OASIS HLB (3 mL, 60 mg, 30 ␮m and 6 mL, 200 mg, 30 ␮m) SPE cartridges were purchased from Waters Corp. (Budapest, Hungary). Strata Si-1 (6 mL, 500 mg, 55 ␮m, 70 A) SPE columns were obtained from Gen-lab (Budapest, Hungary). Strips for pH estimation were purchased from Macherey-Nagel (Düren, Germany). Pig fat samples originated from a Hungarian residue control monitoring program (2010 January to 2010 June) and were stored at −20 ◦ C until analysis. 2.2. Sample clean-up 1.0 g fat sample was weighted into a 50 mL centrifuge tube and 3 mL n-hexane was added to it. Sample was dissolved in n-

Á. Tölgyesi et al. / J. Chromatogr. B 879 (2011) 403–410

hexane by vortex-mixing for 30 s and then was shaken on a Janke & Kunkel IKA KS125 shaker (Staufen, Germany) at 700 min−1 speed for 30 min. Corticosteroids were extracted from the dissolved sample using LLE by adding 4 mL methanol–water mixture (50/50, v/v), followed by vortexing for 1 min, and subsequently shaking again at 700 min−1 for 20 min. After the LLE procedure, the sample was centrifuged at 25 ◦ C for 10 min at 2000 rpm on Sigma 3-18K centrifuge (Osterode am Harz, Germany). The lower layer was transferred to a glass tube and the LLE step was carried out one more time. After centrifuging, the lower layer was transferred again. The two lower layers were combined in the glass tube, homogenized by vortexing, and evaporated under a gentle nitrogen stream at 55 ◦ C in a Caliper TurboVap LV (Hopkinton, MA, USA). The n-hexane layer was discarded. After 30 min, the sample volume was reduced to ∼4 mL, which was cooled down to 25–35 ◦ C at ambient temperature. Five mL distilled water was added to the sample, followed by vortexing for 30 min. The sample was subjected to the SPE procedure, which is described below. An OASIS HLB (3 mL, 60 mg) SPE cartridge was conditioned by passing through 3 mL ethanol, 3 mL methanol, and 3 mL distilled water twice. Sample was applied to the cartridge and allowed to drop down slowly (approximately 0.3 mL/min). SPE column was washed two times with 3 mL distilled water, followed by rinsing twice with 3 mL n-hexane. Cartridge was vacuum dried for 20 s. Sample was eluted with 3 mL acetonitrile. Sample was evaporated to 50–100 ␮L under a gentle nitrogen stream at 45 ◦ C and re-dissolved in methanol–water mixture (50/50, v/v) to a final volume of 500 ␮L. Sample was filtered with a 0.45 ␮m nylon filter (Macherey-Nagel, Düren, Germany) and transferred into a HPLC vial.

2.3. HPLC conditions Corticosteroids were separated using a Supelco Ascentis Express C-18 (150 mm × 4.6 mm, 2.7 ␮m) (Sigma–Aldrich, Budapest, Hungary) Fused-Core analytical column. The separation was carried out using an isocratic method at system backpressure of 300 bar. The mobile phase was a (60/40, v/v) mixture of methanol–acetate buffer (5 mM ammonium acetate and 0.01% acetic acid in water, pH 5.4). The flow rate was 0.8 mL/min and the analysis time was 7.5 min. The injection volume was 10 ␮L and the thermostat of the analytical column was set at 30 ◦ C.

2.4. Instruments and mass spectrometry conditions In the LC–MS/MS system, an Agilent 6410A LC–MS triple quad included an Agilent 1200 HPLC system (G1379A degasser, G1312A binary gradient pump, G1329A auto sampler, and G1316A column thermostat) connected to an Agilent 6410A triple quadrupole (MS/MS) detector, equipped with an Agilent multimode ion source (G1978B) (Agilent Technologies, Palo Alto, CA, USA) was used. Data acquisition was performed using the Agilent Mass Hunter B.01.04 software. Results were evaluated by Agilent Mass Hunter B.01.03 Qualitative and Agilent Mass Hunter B.01.04 Quantitative software. The MS/MS detector was used in the MRM (multiple reaction monitoring) mode for the highest selectivity and sensitivity. The multimode ion source (MMI) was operated in the negative APCI mode. The MS detector settings were as follows: gas temperature—300 ◦ C, gas flow—5 L/min, vaporizer—160 ◦ C, nebulizer pressure—413.7 kPa, capillary voltage—2000 V, and capillary current—4 ␮A.

405

2.5. Quantification Six points (including zero) matrix-matched calibration curve was performed. Blank samples were cleaned-up and after the sample preparation were spiked with different concentration of standard solution to calculate the absolute recovery of the method. Results were evaluated with an internal standard method using linear regressions between relative peak areas and relative concentrations. The internal standard (ISTD) at 20 ␮g/kg cortisol-d4 (Medical Isotopes, Pelham, USA) was added to the samples at the end of sample preparation for correcting the ionization source response and consequently to enhance the accuracy of quantitative determination [20]. 3. Results and discussion 3.1. Optimization of mass spectrometry Corticosteroids give precursor ions in both negative and positive modes [4–6,13,14]. Negative acetate adduct precursor ions have been found more intensive compare to positive [M + H]+ ions. Ammonium acetate is generally used with the ESI source that operates at low temperature. However, ammonium acetate is also suited in the APCI source because the vaporizer temperature of the adduct ions in the MMI is only 160 ◦ C. Initially, the acetate adduct precursor ions were optimized using individually diluted 1 ␮g/mL methanolic standard solutions in the MS2 scan mode. The mass spectra were recorded at different fragmentor voltage ranging from 90 to 150 V in order to find the highest response for the chosen precursor ions. Ion transitions were optimized using a product ion scan mode. Precursor ions were fragmented on different collision energies (CE) between 0 and 30 V. The two most intensive ion traces were chosen on CEs that gave the highest response. In the MRM mode, the most intensive ion transition was used for quantification. Qualification was performed using other ion transitions. ISTD was identified using one ion trace (Table 2). 3.2. General conditions for liquid chromatography Fat contains several non-polar compounds such as mono-, di, and tri fatty acid esters of glycerol, which could not be removed completely from corticosteroids in a clean up procedure. An analytical column (Ascentis Express), which enables high peak capacity, was tested in both gradient and isocratic modes [6,17]. In the previous study, a peak capacity of ∼150 was attainable on a small (50 mm × 2.1 mm, 2.7 ␮m) Ascentis Express C-18 column with an analysis time of 25 min [17]. In our study, we used a longer column (150 mm × 4.6 mm, 2.7 ␮m) to avoid column overload and to get the highest resolution between lipophilic matrices and corticosteroids. A fully porous column Phenomenex Gemini C-18 (150 mm × 4.6 mm, 5 ␮m) (Gen-lab, Budapest, Hungary) was also tested, but the chromatographic peaks were narrower on shell-type column than on the porous column. The sensitivity in using Ascentis Express was also enhanced. Isocratic separation gave the highest resolution between matrices and steroids because it is generally more selective for the structurally related compounds compare to the gradient elution. Significantly, the isocratic separation reduced the analysis time to 7.5 min, which is three and two times faster than analysis conducted using Gemini C-18 column in a gradient mode and Zorbax Eclipse XDB (100 mm × 2.1 mm, 1.8 ␮m) column in an isocratic separation, respectively [5,14]. In order to elute very non-polar compounds possibly concentrated on the inlet of the column during the isocratic elution with the methanol–acetate buffer (60/40, v/v) mobile phase, the column was washed with methanol–acetate buffer (95/5, v/v) at 0.8 mL/min flow rate for

(11.5–21.3) ± 1.2 16.4 ± 1.2 90V

110 V

90 V

140 V

90 V

110 V

75 ms 75 ms 75 ms 75 ms 75 ms 75 ms 75 ms 75 ms 75 ms 75 ms 75 ms 329.4 280.1 342.2 313.2 379.2 305.3 361.1 307.2 343.2 309.2 335.1 4.0–7.5 2

Cortisol-d4

MET-PRED

DXM

FLU

400 MRM

APCI

negative

MET-PREDON

[M+CH3 COO]− 419.3 [M+CH3 COO]− 431.2 [M+CH3 COO]− 469.3 [M+CH3 COO]− 451.3 [M+CH3 COO]− 433.2 [M+CH3 COO]− 425.3 data not stored PRED 0 APCI 0–4.0 1

Ion ratios of compounds in standard solutions were calculated as an average of the five calibration points and the ratios of spiked samples were calculated from the 54 fortified samples (3days, 3 levels and 6 parallel). EMV = Delta Electron Multiplier Voltage.

(18.1–30.1) ± 1.4 24.1 ± 1.4

13.8–18.3

19.0–30.0

(9.7–17.9) ± 1.3 13.8 ± 1.3

9.6–16.8

(13.7–25.4) ± 1.2 19.5 ± 1.2

5.4–12.3 (3.7–11.1) ± 1.6 7.4 ± 1.6

20 V 25 V 5V 15 V 10 V 30 V 15 V 30 V 15 V 25 V 15 V

Maximum permitted tolerances Ion ratios of standard solutions FragmentorCE Dwell time Product ions Precursor ion Compound EMV Ion Polarity Ion Mode Scan Type Time (min) Segment

Table 2 Agilent 6410A MS/MS equipped with multimode ion source: settings and ion ratios of two ion transition reactions of the analytes in standard solutions and spiked samples.

17.7–23.7

Á. Tölgyesi et al. / J. Chromatogr. B 879 (2011) 403–410

Ion ratios of spiked samples

406

9 min for every six injections. This washing was important to avoid column overload during the validation. Cortisol-d4 as an ISTD was applied successfully for pig fat samples. Because cortisol-d4 had lower intensity in MS analysis in comparison with detected compounds, a concentration of 20 ␮g/kg was used, which was equal to the highest calibration point of METPREDON and METPRED. This ISTD concentration was determined to be good for analysis. In method development of liquid chromatography, the first step of making decision is the pH control of mobile phase. Since corticosteroids have no ionic group in their structures, pH adjustment is not necessary. However, in the MS/MS detection, the acetate concentration of the mobile phase could increase the detector responses due to the acetate adduct precursor ions. Hence, the acetate concentration of the mobile phase was optimized using four different acetate buffers at different pH. All buffer solutions contained 5 mM ammonium acetate, but different percentages of acetic acid: solution 1 did not contain acid (pH 7.0), solution 2 contained 0.01% (v/v) acid (pH 5.4), solution 3 contained 0.025% (v/v) acid (pH 4.4) and solution 4 contained 0.05% (v/v) acid (pH 4.1). The mobile phases were mixture of methanol and different acetate buffers (60/40, v/v). A cleaned blank sample was spiked with 4 ␮g/kg PRED, 10 ␮g/kg METPREDON, 1 ␮g/kg FLU, 1 ␮g/kg DXM, 10 ␮g/kg METPRED, and 20 ␮g/kg cortisol-d4 and then injected into the different mobile phases. A standard solution that contained the same standards at the same concentration levels in methanol–water (50/50, v/v) was also injected before the fortified sample. Peak areas were integrated in all conditions. Fig. 1A and B show the absolute areas of standards. As can be seen, 0.01% (v/v) acetic acid in 5 mM ammonium acetate solution had the maximum intensity for ion traces of both spiked sample and standard solutions. 3.3. Optimization of sample preparation 3.3.1. Optimization of NP SPE clean-up Nine 1.0 g blank pig fat samples, three each from three different sources, were weighted into 50 mL centrifuge tubes. All samples were spiked with 4 ␮g/kg PRED, 10 ␮g/kg METPREDON, 1 ␮g/kg FLU, 1 ␮g/kg DXM, 10 ␮g/kg METPRED prior to dissolving them in different solvents. Three samples each were dissolved separately in 6 mL of n-hexane, n-hexane–dichloromethane (50/50, v/v) mixture, and dichloromethane. Samples were directly cleaned and concentrated on Strata Si-1 (6 mL, 500 mg) cartridges. SPE columns were conditioned with 6 mL organic solvents, which were similar to the sample solvents (n-hexane, n-hexane–dichloromethane (50/50, v/v), and dichloromethane). Samples were poured into the cartridges at a flow rate of ∼0.3 mL/min. Cartridge was rinsed twice with 6 mL washing solutions that were the same as condition solvents. Columns were vacuum dried for 1 min and samples were eluted with 6 mL ethyl acetate. A 20 ␮g/kg ISTD was added to the prepared samples, which were evaporated under a gentle nitrogen stream at 45 ◦ C and re-dissolved in methanol–water (50/50, v/v) mixture to the final volume of 500 ␮L. Samples were filtered into HPLC vials for injecting to the LC–MS/MS. Recovery results were evaluated by comparing the corticosteroids’ relative areas to standard solutions’ relative areas that were prepared in clear methanol–water (50/50, v/v) mixture to learn different conditions. Conditions and recovery results (mean concentration and precision) are summarized Table 3. The recovery results were similar under different conditions, however, the precision (RSD%) was improved with increase in the strength of the washing solvent with dichloromethane (Table 3). n-hexane could not wash out the absorbed polar matrices, but increasing the polarity with dichloromethane showed improved reproducibility. However, the recovery was not satisfactory for all corticosteroids (50.3–87.0%) (Table 3).

104.9% (8.4%) 101% (3.2%) 103% (22.3%) 104% (7.9%) 101.6% (5.4%) 3 mL ACN

91.7% (25.9%) 95% (22.3%) 75% (18.7%) 105.8% (20%) 108% (27.2%) 3 mL ACN

94.5% (55%) 102% (30.7%) 103% (32.8%) 90% (11.5%) 90% (16.1%) 5 mL ACN

103.7% (4.6%) 89% (6.7%) 71% (1.6%) 93.7% (6.1%) 93% (2.8%) 6 mL ACN

104% (1.7%) 85% (7.2%) 69% (4.4%) 95.3% (1.2%)

95.7% (9.5%) 84% (4.4%) 65% (8.6%) 80.7% (1.9%) 84% (2.3%)

89.7% (4.2%) 6 mL ACN

water/metanol (50/50, v/v) water/ACN (50/50, v/v) ACN

water/ACN (50/50, v/v) water/metanol (50/50, v/v)

3 mL n-hexane

3 mL n-hexane

3 mL n-hexane

water 3 mL n-hexane

3 mL n-hexane

–

3 mL n-hexane

–

6 mL DCM/n-hexane (50/50, v/v) 6 mL DCM

DCM = dichloromethane; ACN = acetonitrile; ETAC = ethyl acetate.

6 mL ETAC

6 mL ACN

2 × 6 mL water followed by 2 × 6 mL n-hexane 2 × 6 mL water followed by 2 × 6 mL n-hexane 2 × 6 mL water followed by 2 × 6 mL n-hexane 2 × 6 mL water followed by 2 × 6 mL n-hexane 2 × 3 mL water followed by 2 × 3 mL n-hexane 2 × 3 mL water followed by 2 × 3 mL n-hexane

50.3% (6.6%) 87% (1.9%) 87% (10%) 81.1% (4%)

72.5% (5.2%) 6 mL ETAC

2 × 6 mL DCM/n-hexane (50/50, v/v) 2 × 6 mL DCM

50.3% (15.5%)

METPREDON

74% (14.8%) 52.7% (14%)

PRED Elution solvent

6 mL ETAC

Washing solution

2 × 6 mL n-hexane

Strata-Si-1 (6 mL, 500 mg) Strata-Si-1 (6 mL, 500 mg) Strata-Si-1 (6 mL, 500 mg) HLB (6 mL, 200 mg) HLB (6 mL, 200 mg) HLB (6 mL, 200 mg) HLB (6 mL, 200 mg) HLB (3 mL, 60 mg) HLB (3 mL, 60 mg) – 6 mL n-hexane

SPE LLE (2 × 4 mL) Sample solvent

3.3.2. Optimization of RP SPE clean-up Samples were initially subjected to the RP SPE clean-up. They were dissolved in 3 mL n-hexane and extracted into different solvents (LLE), followed by cleaned-up on small (3 mL, 60 mg) and on big (200 mL, 60 mg) HLB SPE cartridges. Three different pig fat samples in four parallel were spiked with 4 ␮g/kg PRED, 10 ␮g/kg METPREDON, 1 ␮g/kg FLU, 1 ␮g/kg DXM, 10 ␮g/kg METPRED. Three samples each were extracted with 4 mL water, 4 mL acetonitrile–water mixture (50/50, v/v), 4 mL methanol–water mixture (50/50, v/v), and 4 mL acetonitrile. Conditions are summarized in Table 3. The cleaning procedure was the same as described in Section 2.2. When samples were extracted with 100% acetonitrile, the extract was evaporated to 0.5 mL and was re-dissolved in 9 mL water. Aquatic extract was not evaporated. First the different LLEs were tested using bigger HLB for SPE to avoid the column overload. Applying 100% water for LLE was good; suggesting feasibility of selective extraction of corticosteroids from the non-polar matrix with water. However, with increase in the organic phase in an extraction solvent with methanol or acetonitrile, the extraction efficiency improved (Table 3). Acetonitrile (100%) gave the best recovery, but it had low precision. Organic phase increased the extraction of some non-polar matrices, which might have influenced the analysis. All in all, LLE with methanol–water or acetonitrile–water mixture (50/50, v/v) resulted in the best recovery and precision (Table 3).

Table 3 Conditions and results of different LLE and SPE methods. Recovery rates % (n = 3) (RSD%).

Fig. 1. A standard solution, which contained 4 ␮g/kg PRED, 10 ␮g/kg METPREDON, 1 ␮g/kg FLU, 1 ␮g/kg DXM, 10 ␮g/kg METPRED and 20 ␮g/kg cortisol-d4 in clear HPLC solution, was injected into mixture of methanol–acetate buffer (60/40, v/v) that contained different acetate buffer (A). A cleaned-up blank sample, which was spiked after the preparation with 4 ␮g/kg PRED, 10 ␮g/kg METPREDON, 1 ␮g/kg FLU, 1 ␮g/kg DXM, 10 ␮g/kg METPRED and 20 ␮g/kg cortisol-d4, was also injected into different mobile phases (B). All buffers contained 5 mM ammonium acetate, but different percentage of acetic acid. Solution 1: did not contained acid (pH 7.0), solution 2: contained 0.01% (v/v) acid (pH 5.4), solution: 3 contained 0.025% (v/v) acid (pH 4.4) and solution 4: contained 0.05% (v/v) acid (pH 4.1). The columns show the detector responses.

53.3% (19.8%)

42% (10.8%) 70% (15.2%) 101% (10.1%)

47.6% (15.9%) 70% (21.4%)

407

FLU

104% (23.1%)

DXM

METPRED

Á. Tölgyesi et al. / J. Chromatogr. B 879 (2011) 403–410

−6.8 −1.7 −1.9 0.0 −4.3 8.2 −3.2 4.9 5.2 5.5 −0.5 −7.3 0.1 6.8 −1.4 15.5 −8.7 7.1 12.4 8.7 −10.7 7.7 −9.4 −3.7 8.3 3.6 6.2 −3.1 1.3 19.4 −14.0 −11.7 −17.4 −18.0 −17.4 −0.2 −13.0 −11.7 −13.8 −8.9 matrix effect % 13.7 5.4 7.4 13.5 26.9 [(SS1/RS) − 1] × 100 [(SS2/RS) − 1] × 100 [(SS3/RS) − 1] × 100 [(SS4/RS) − 1] × 100 [(SS5/RS) − 1] × 100

−2.0 7.0 0.4 7.9 15.1

1.5885 1.4810 1.5616 1.5583 1.5881 1.5200 2.7 27665 29927 26792 29034 29098 29187 4.1 0.2265 0.2254 0.2099 0.2266 0.2420 0.2233 5.1 3944 4555 3601 4223 4434 4288 8.8 0.2611 0.2332 0.2813 0.2365 0.2514 0.2828 9.3 4547 4712 4827 4406 4606 5430 8.1 1.2688 1.0916 1.1205 1.0476 1.0401 1.0485 3.3 22096 22059 19225 19518 19058 20135 6.1 0.5668 0.5554 0.6066 0.5691 0.6116 0.6524 6.4

Absolute areas (cps) Relative areas

Absolute areas (cps)

Relative areas

Absolute areas (cps)

Relative areas

METPRED DXM FLU

Absolute areas (cps)

9871 11223 10408 10603 11207 12527 7.4

Selectivity was proven by analyzing ten different pig fat samples. Fig. 2 shows the TIC (total ion chromatogram) of a spiked fat sample. There were no any interfering peaks in blank TIC sample where corticosteroids were eluted (Fig. 2).

RS SS1 SS2 SS3 SS4 SS5 RSD% (n = 5)

3.6. Selectivity

Relative areas

For the confirmation of banned and MRL substances, a minimum of four and three identification points shall be required [16]. In MS/MS detection, one precursor ion refers to one identification point and one ion transition adds 1.5 point. One precursor ion and its two ion transitions (quantify and qualify ion traces) were used for an analyte that gives the four identification points. The intensity ratio of the quantifier and qualifier transitions was also seen in standard solution and in samples as well. The EU guideline determines the acceptable range of qualifier ratios in samples [16]. The qualifier ratios in samples were calculated from 54 samples and were in the satisfied range (Table 2).

Absolute areas (cps)

3.5. Identification

METPREDON

The absolute matrix effect (ME) was quantified using an earlier reported satisfied method, ME% = [(area of SS)/(area of RS) − 1] × 100 [18]. ME = 0 means there is no ion effect during the analysis, ME > 0 shows ion enhancement and ME < 0 means ion suppression. Five blank fat samples that originated form different sources were cleaned-up using the optimized sample preparation procedure. Samples were fortified after the preparation with 4 ␮g/kg PRED, 10 ␮g/kg METPREDON, 1 ␮g/kg FLU, 1 ␮g/kg DXM, 10 ␮g/kg METPRED, and 20 ␮g/kg cortisol-d4 (spiked sample, SS). A reference solution (RS) containing the same standards in the same concentrations was prepared in methanol–water (50/50, v/v). Samples were injected into the LC–MS/MS instrument. Areas were integrated using both an external standard method (absolute areas) and an internal standard method (relative areas). Signal values and RSD% are summarized in Table 4. In the case of PRED, DXM, and METPRED the ISTD could well compensate the ionization source response. The ME was found better (they were nearer to zero) using cortisol-d4 as ISTD in the evaluation. The RSD% of relative areas was also lower compare to absolute areas (Table 4). For METPREDON, an internal standard method resulted in lower RSD% for relative peak areas, but ME was better using an external method (Table 4). In the case of FLU, the evaluation with external standard method was found to be better. However, cortisol-d4 was used with success for both METPREDON and FLU because it improved the recovery during the validation procedure.

PRED

3.4. Matrix effect and choice of internal standard

Sample

Clean-up procedure was also tested on small (3 mL, 60 mg) HLB SPE cartridges using acetonitrile–water and methanol–water (50/50, v/v) mixtures for LLE. When acetonitrile–water mixture was applied, the recoveries were similar between small and big HLB SPE (75–108% and 71–103.7%, respectively), but the precision of larger HLB SPE was found better than smaller SPE (Table 3). Surface area thus influenced the results, which could also be seen when methanol–water (50/50, v/v) mixture was applied. Exception was FLU that had significantly higher RSD% (22.3%) using methanol–water mixture for LLE, but the precisions of other corticosteroids were satisfied (Table 3). However, recoveries improved in the use of smaller cartridge (101–104.9%) than larger HLB (69–104%) (Table 3). Methanol–water (50/50, v/v) mixture for LLE and subsequent HLB SPE clean-up on small cartridge provided the best conditions for cleansing pig fat samples for LC–MS/MS analysis.

Relative areas

Á. Tölgyesi et al. / J. Chromatogr. B 879 (2011) 403–410 Table 4 Detector responses (absolute and relative areas) of a reference standard (RS) at MRL or mrpl level, respectively in clear HPLC solution and in five spiked samples (SS) that originated from different sources. Samples were spiked after the preparation with standards to MRL or mrpl values, respectively and a 20 ␮g/kg ISTD was added to them. Precisions (RSD%) were calculated from the spiked samples’ areas (n = 5). Matrix effect ME% = [(area of SS)/(area of RS) − 1] × 100. ME = 0 means there is no ion effect during the analysis, ME > 0 shows ion enhancement and ME < 0 means ion suppression.

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Table 5 Recovery (n = 18 per a level) (mean% and range%) at 0.5 MRL, MRL, 1.5 MRL and mrpl, 1.5 mrpl, 2 mrpl and within-laboratory reproducibility (n = 18 per a level). 0.5MRL

PRED MET-PREDON FLU DXM METPRED

MRL/mrpl

1.5MRL/1.5mrpl

Mean recovery%

Range%

RSD%

Mean recovery%

Range%

RSD%

89 92

71–108 77–115

10.1 11.8

89 98 87 81 89

77–100 81–119 71–112 65–109 78–106

8.4 11.6 10.7 20.5 8.9

83

74–97

8.0

Mean recovery% 91 100 93 93 91

2mrpl Range%

RSD%

81–105 82–120 79–110 74–120 78–104

7.0 12.3 9.9 12.4 8.4

Mean recovery%

Range%

RSD%

92 92

84–105 72–113

6.5 12.7

Table 6 Decision limit (CC␣) and detection capability (CC␤), LOD and LOQ.

PRED METPREDON FLU DXM METPRED

Fig. 2. Total ion chromatogram (TIC) of a spiked fat sample (2 ␮g/kg PRED, 5 ␮g/kg METPREDON, 1 ␮g/kg FLU, 1 ␮g/kg DXM, 5 ␮g/kg METPRED) and a blank one (under).

3.7. Linearity Calibration was based on the matrix matched curve. Prepared blank samples were spiked with different concentrations of standards and with 20 ␮g/kg ISTD after the clean-up. Six-point calibrations (including zero) were performed between 0 and 8.0 ␮g/kg for PRED and 0 and 20.0 ␮g/kg for METPREDON and METPRED. In the case of banned substances (FLU and DXM), calibrations ranged from 0 to 4.0 ␮g/kg. Correlation coefficients (r2 ) ranged between 0.9893 and 0.9997. 3.8. Recovery, within-laboratory reproducibility Samples were fortified with the standard solution on three levels in six series. For MRL substances (PRED, METPREDON and METPRED) the spiking levels were on 0.5 MRL, MRL and 1.5 MRL. Concentrations used were 2.0, 4.0 and 6.0 ␮g/kg for PRED and 5.0, 10.0 and 15.0 ␮g/kg for METPREDON and METPRED. The fortifying level was mrpl, 1.5 mrpl and 2.0 mrpl (1.0, 1.5 and 2.0 ␮g/kg, respectively) for mrpl compounds. These levels meet the 2002/657/EC Decision [2,16]. Recovery measurements were repeated for additional two days by different operators, solvents, and batch of SPE cartridges (within-laboratory reproducibility). The absolute recovery results were calculated from 18 results per a level and were found in the range of 81–100% for corticosteroids (Table 5). These results satisfied the EU guideline [16]. The within-laboratory reproducibility means the precision of method [16]. Precision is expressed as the relative standard deviation (RSD%) of the method [19]. The within-laboratory reproducibility was found in the range from 6.5 to 12.3% for all corticosteroids, expect DXM. The precision for DXM was in the range from 12.7 to 20.5% (Table 5). The within-laboratory reproducibility was also acceptable [16]. 3.9. Decision limit (CC˛) and detection capability (CCˇ), limit of detection (LOD) and limit of quantification (LOQ) The decision limit (CC␣) means the limit at and above which it can be concluded with an error probability of ˛ that a sample is non-compliant. In the case of PRED, METPRED and METPREDON with established MRL (˛ = 5%), CC␣ was calculated as MRL plus 1.64 times the standard deviation of the within-laboratory repro-

CC␣ (␮g/kg)

CC␤ (␮g/kg)

LOD (␮g/kg)

LOQ (␮g/kg)

4.5 11.9 0.2 0.1 11.3

5.0 13.8 0.30 0.18 12.6

0.3 0.3 0.2 0.1 0.2

1.0 1.0 0.66 0.33 0.66

ducibility at the MRL level [16]. For substances having no MRL (˛ = 1%), twenty blank matrix samples from ten different sources were analyzed to calculate the signal-to-noise ratio (S/N) at the time window in which the analyte is expected. CC␣ values were defined as three times of S/N (Table 6). The detection capability (CC␤) was calculated as the value of the decision limit plus 1.64 times the standard deviation of the within-laboratory reproducibility at the decision limit [16]. For banned substances, CC␣ and CC␤ were found lower than mrpl and decision limits were confirmed by analyzing twenty different blank fat samples which were fortified to the calculated concentrations, which satisfy the 2002/657/EC [2,16]. Limit of detection (LOD) was calculated as 3 times of S/N and limit of quantification (LOQ) as 10 times of S/N (Table 6). LODs were also confirmed as the decision limits. Decision limits and LODs were accepted for a compound when the signal-to-noise ratios for spiked samples were higher than three and the ion ratios were in an acceptable range. 4. Conclusions A simple and fast LC–MS/MS method was developed for qualify and quantify corticosteroids in pig fat samples, which include LLE with methanol–water (50/50, v/v), SPE clean-up on HLB cartridge for the sample preparation, and isocratic separation on Ascentis Express HPLC column using a (60/40, v/v) mixture of methanol–5 mM ammonium acetate buffer containing 0.01% acetic acid, pH 5.4. An addition of cortisol-d4 as an internal standard at the end of the sample preparation was suitable for studied corticosteroid in order to correct the ionization source response that improved the recovery and precision of the method. Method was successfully validated in accordance with EU 2002/657/EC Decision. References [1] Commission Regulation (EU) 37/2010, Off. J. EU. Legis (2010) L 15/1. [2] P. Gowik, J. Chromatogr. A 1216 (2009) 8051. [3] EMEA/MRL/798/01-FINAL, Committee for veterinary medicinal products, Methylprednisolone, Summary report (2), http://www.ema.europa.eu/pdfs/vet/mrls/079801en.pdf. [4] Á. Tölgyesi, Z. Verebey, V.K. Sharma, L. Kovacsics, J. Fekete, Chemosphere 78 (2010) 972. [5] Á. Tölgyesi, V.K. Sharma, L. Kovacsics, J. Chromatogr. B 878 (2010) 1471. [6] Á. Tölgyesi, L. Tölgyesi, V.K. Sharma, M. Sohn, J. Fekete, J. Pharm. Biomed. Anal. 53 (2010) 919. [7] I. Franco, M.C. Escamilla, J. García, M.C.G. Fontán, J. Carballo, J. Food Compos. Anal. 19 (2006) 792.

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