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Liquid chromatographic determination of levamisole in animal plasma: ultraviolet versus tandem mass spectrometric detection
Analytica Chimica Acta 483 (2003) 215–224
Liquid chromatographic determination of levamisole in animal plasma: ultraviolet versus tandem mass spectrometric detection Siegrid De Baere∗ , Marc Cherlet, Siska Croubels, Kris Baert, Patrick De Backer Departments of Pharmacology, Pharmacy and Toxicology, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, B-9820 Merelbeke, Belgium Received 2 July 2002; received in revised form 1 October 2002; accepted 22 October 2002
Abstract This work presents the optimization and validation of a liquid chromatography (LC)–ultraviolet (UV) detection method and a tandem mass spectrometry (LC–MS/MS) method with positive electrospray ionization for the determination of levamisole, an anthelmintic for veterinary use, in animal plasma. A liquid–liquid extraction procedure in alkaline medium, using hexane-isoamyl alcohol (95:5, v/v) as extraction solvent, was performed to clean-up the plasma samples prior to LC-UV analysis. The sample preparation prior to LC–MS/MS analysis consisted of a protein precipitation step, using acetonitrile. Methyllevamisole was used as the internal standard. Chromatographic separation was achieved on a LiChrospher® 60 RP-select B (5 m) column using a mixture of 0.1 M ammonia acetate in water and acetonitrile as the mobile phase. A gradient (flow rate 1 ml min−1 ) or isocratic (flow rate 0.2 ml min−1 ) elution was performed in case of the LC-UV and LC–MS/MS methods, respectively. UV detection was at 235 nm. The mass spectrometer was operated in the multiple reaction monitoring (MRM) mode. The methods were validated (linearity, precision, trueness, limit of quantification, limit of detection, specificity) according to the requirements defined by the European Community. Good linearity was achieved over the concentration ranges tested (0–0.5 g ml−1 and 0–4.0 g ml−1 , r > 0.99, goodness-of-fit <10%). Limits of quantification of 0.025 g ml−1 and 0.1 g ml−1 were achieved for the LC–MS/MS and LC-UV methods, respectively. The limits of detection were 0.009 and 0.077 g ml−1 , respectively. The results for precision (within-day and between-day) fell within the ranges specified. The methods have been successfully used for the determination of levamisole in plasma samples from two pigs medicated via drinking water. A good correlation was observed between the results of both methods (r 2 = 0.9831, slope = 1.13, intercept = 0.005 g ml−1 ), proving their usefulness for the application in the field of pharmacokinetic and residue analysis. © 2002 Elsevier Science B.V. All rights reserved. Keywords: Levamisole; Anthelmintic; Animal plasma; Liquid chromatography-ultraviolet; Liquid chromatography–mass spectrometry/MS; Tandem mass spectrometry; Validation; Pharmacokinetics; Residue analysis
1. Introduction Levamisole, (S)-(−)-2,3,5,6-tetrahydro-6-phenylimidazo-[2,1b]thiazole (LEV, Fig. 1) is a potent broad-spectrum anthelmintic drug, which is widely ∗ Corresponding author. Tel.: +32-9-264-73-24; fax: +32-9-264-74-97. E-mail address: [email protected] (S. De Baere).
used in veterinary medicine for the control of gastrointestinal parasites in cattle, sheep and pigs. It is normally administered orally, by pour-on or by subcutaneous or intramuscular injection. The recommended dose is 8 mg kg−1 body weight (BW). Various techniques have been used for the determination of levamisole in biological fluids. They include gas chromatography with nitrogen–phosphorus detection (GC-NPD) [1,2] and flame ionization detection
0003-2670/03/$ – see front matter © 2002 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 3 - 2 6 7 0 ( 0 2 ) 0 1 3 7 7 - 6
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Fig. 1. Chemical structures and tandem mass spectra of levamisole and methyllevamisole obtained after direct infusion of standard solutions of 10 g ml−1 of both components.
(GC-FID) [3] and liquid chromatography (LC) with ultraviolet (UV) detection [4–6]. No GC–mass spectrometry (GC–MS) or LC–MS methods have been reported for the determination of levamisole in plasma. For the GC methods, the clean-up of the plasma samples consists of a rather time consuming liquid–liquid back extraction procedure, consecutively in alkaline, acidic and alkaline media [1–3]. Loussouarn et al. also described a solid-phase extraction (SPE) procedure using octyl (C8) cartridges [2]. The sample preparation for the LC methods is generally less complicated and consists of a liquid–liquid extraction procedure in alkaline medium [5,6] or a SPE procedure using octadecyl (C18) columns. Gas chromatography is performed on glass columns packed with 3% OV-17 on Supelcoport [1,3] or Gas-Chrom Q (80–100 mesh) [2]. Reversed-phase ion-pair chromatography is applied for the elution of levamisole from the LC columns. Ultraviolet detection was performed at 225 nm. Some authors mention the use of an internal standard (IS, e.g. methyllevamisole [1]; trimipramine [3]; quinine [6]) in order to ameliorate the quantification of levamisole. The reported limit of quantification (LOQ) and limit of detection (LOD) values are generally lower for the GC than for the LC methods (GC—LOD: 10 ng ml−1 [1,2], LOQ: 1.9 g ml−1 [3]; LC-UV— LOD: 21–50 ng ml−1 [4,6], LOQ: 72–80 ng ml−1 [5,6]).
The purpose of this work was to develop a method for the determination of levamisole in animal plasma which could be used for pharmacokinetic and residue studies with animals. Therefore, a LC-UV method was optimalized in the first instance. Because many samples have to be analyzed in pharmacokinetic studies, special attention has been paid to enhanced speed of analysis, without loss of sensitivity and specificity. Hence, a LC–MS/MS method was developed subsequently. Both methods were fully validated according to the requirements defined by the European Community (linearity, precision and accuracy, LOQ and LOD, specificity) [7]. The practical applicability of the methods was evaluated during the routine analysis of plasma samples which were obtained during a pharmacokinetic and residue study in pigs after the administration of levamisole via drinking water (dose level: 8 mg kg−1 body weight; administration period: 8 h).
2. Experimental 2.1. Standards and chemicals Levamisole hydrochloride was a chemical reference substance (CRS) of the European Pharmacopoeia (Strasbourg, France). A stock solution of 1000 g ml−1 was prepared in methanol. It was
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stored at ≤−15 ◦ C and replaced by a fresh stock solution every 3 months. Working solutions at levamisole concentrations of 100, 80, 60, 40, 20, 10 and 5 g ml−1 (calibration graph A) and 10, 8, 6, 4, 2, 1 and 0.5 g ml−1 (calibration graph B) were prepared by appropriate dilution of the stock solution with water. By spiking 1 ml (LC-UV method) or 500 l (LC–MS/MS method) of plasma with 50 or 25 l of each working solution, respectively, calibration graphs ranging from 0.25 to 4 g ml−1 (calibration graph A) and 0.025 to 0.5 g ml−1 (graph B) were obtained. All the working solutions were stored in the refrigerator (2–8 ◦ C) and were replaced by a fresh solution every month. Methyllevamisole hydrochloride (Fig. 1), used as the (IS) was purchased from Janssen Pharmaceutica (Beerse, Belgium). A stock solution of 1000 g ml−1 methyllevamisole was prepared in methanol. Two working solutions at concentrations of 40 and 4 g ml−1 were prepared by diluting the stock solution with water. Storage conditions were the same as for the levamisole stock and working solutions. All solvents used for the mobile phase (acetonitrile, tetrahydrofuran (THF) and water) were of LC grade and were purchased from Acros (Geel, Belgium). All other products and solvents used for the preparation of stock solutions and for extraction were of analytical reagent grade and were purchased from Merck (Darmstadt, Germany—methanol, hexane, isoamyl alcohol, triethylamine and sodium hydroxide pellets) and from Sigma (Bornem, Belgium—ammonium acetate). 2.2. Biological samples Known levamisole-free plasma samples were obtained from pigs which did not receive any medication. Incurred plasma samples were obtained from two pigs which received levamisole via the drinking water at a dose rate of 8 mg kg−1 body weight during an 8 h period. Blood samples were taken before the start of drug administration and at regular time intervals after the end of the medication period (0, 0.5, 1, 2, 4, 7, 16, 24 and 40 h) in identified heparinized tubes. All samples were centrifuged within 1 h after sampling and the plasma was transferred in identified plastic tubes. The samples were stored at ≤−15 ◦ C pending analysis.
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2.3. Apparatus LC-UV system: The LC-UV system consisted of a ternary gradient pump Model 9012, an autosampler Model 410 ProStar and a DAD detector Model ProStar, all from Varian (Walnut Creek, CA). Detection was performed at 235 nm. Quantification was done using the STAR software (Varian). LC–MS/MS system: The LC system consisted of an Alliance type 2690 Separations and a Column Heater Module, both from Waters (Milford). The LC column effluent was pumped to a Quattro Ultima triple quadrupole mass spectrometer (Micromass, Manchester, UK), equipped with an electrospray ionization (ESI) Z-spray source, which was used in the positive ion mode. Quantification was done with the Masslynx software (Micromass) using the mentioned multiple reaction monitoring (MRM) transitions. 2.4. Sample preparation procedure LC-UV method: One milliliter of plasma was transferred into a Pyrex extraction tube and 50 l of the appropriate (IS) working solution was added (40 or 4 g ml−1 , depending on the expected levamisole concentration in the sample). After vortex mixing for 15 s, 2 ml of 1 M sodium hydroxide and 4 ml of the extraction solvent (hexane/isoamyl alcohol, 95:5, v/v) were added and the samples were extracted by rotation (using a home-made mechanical rotation apparatus) for 10 min, followed by a 10-min centrifugation step (2500 rpm, Jouan type C412, Vel, Leuven, Belgium). Thereafter, the organic phase was transferred into a small pyrex tube and evaporated under a gentle stream of nitrogen gas at 40–45 ◦ C (Pierce Reacti-ThermTM heating module and evaporating unit type 18,780, Vel). The dry residue was reconstituted in 250 l of mobile phase A, vortex mixed for 15 s and transferred into an autosampler vial. A 100 l aliquot was injected into the LC-UV system. LC–MS/MS method: Five hundred microliters of plasma was transferred into an Eppendorf centrifuge tube and 25 l of the appropriate IS working solution (40 or 4 g ml−1 , depending on the concentration expected in the incurred samples) was added. After vortex mixing for 15 s, 500 l of acetonitrile was added, followed by a 15 s vortex-mixing step and a 10 min centrifugation step (10,800 rpm, Abbott centrifuge,
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Abbott Diagnostics Division, Belgium). The supernatant was transferred into an autosampler vial and a 25 l aliquot was injected into the LC–MS/MS system.
For our quantitative purposes, the instrument was operated in the MRM mode, using for each analyte one protonated molecular ion > product ion transition: m/z = 205.1 > m/z = 178.2 for levamisole and m/z = 219.2 > m/z = 192.2 for the IS.
2.5. Chromatography 2.7. Method validation LC-UV system: Chromatographic separation was achieved using a reversed-phase Lichrospher® RP-select B (5 m) column (125 mm × 4 mm i.d.), in combination with a guard column of the same type (4 mm × 4 mm i.d.), from Merck. The mobile phase A was a solution of 0.1 M ammonium acetate in water, containing 7.7% (v/v) of tetrahydrofuran and 0.3% (v/v) of triethylamine, while the mobile phase B was pure acetonitrile. A gradient elution was performed: 0–7 min: 65% A, 35% B; 7 to 8 min: linear gradient from 65% A to 50% A; 8–14 min: 50% A, 50% B; 14 to 15 min: linear gradient from 50% A to 65% A; 15–20 min: 65% A, 35% B [8]. Eluate was delivered to the LC column at 1.0 ml min−1 . LC–MS/MS system: Chromatographic separation was achieved using the same type of column and pre-column as for the LC-UV system. The composition of the mobile phase solvents A and B was also the same as those used for the LC-UV system. Mobile phase was delivered to the LC column at 0.2 ml min−1 . An isocratic elution was performed (0–10 min: 60% A, 40% B). 2.6. Mass spectrometry The instrument was calibrated with a solution of sodium iodide according to the manufacturer’s instructions. Thereafter, tuning was performed for the analytes of interest by direct infusion of a 1 g ml−1 solution at 9 l min−1 using a Hamilton syringe (200 l, Bonadoz, Switzerland) and a Harvard pump 11 apparatus (Holliston, USA). The following tuning parameters were used: capillary: 2.8 kV, cone: 30 V, source temperature: 120 ◦ C, desolvation temperature: 250 ◦ C, cone gas flow: ±30 l h−1 , desolvation gas flow: ±600 l h−1 , resolution (LM1, HM1, LM2, HM2): 15.0, ion energy 1:2.0, ion energy 2:0.5, entrance: 20, exit: 15, multiplier: 650 V, Pirani pressure: ±2.9 × 10−3 mbar, dwell time 0.5 s. The optimal settings for collision energy were different for levamisole and the IS, i.e. 24 and 20 eV, respectively.
The proposed methods for the determination of levamisole in animal plasma were validated by a set of parameters which are in compliance with the recommendations as defined by the European Community and with our own criteria which were based on the literature [7]. The linearity of the methods was evaluated by analyzing calibration samples, which were prepared by spiking blank plasma from pigs that did not receive any medication with levamisole. The addition of 50 l (LC-UV method) or 25 l (LC–MS/MS method) of the above mentioned standard working solutions resulted in analyte concentrations of 0.25, 0.5, 1.0, 2.0, 3.0 and 4.0 g ml−1 (calibration A) and 0.025, 0.05 (only for the LC–MS/MS method) 0.1, 0.2, 0.3, 0.4 and 0.5 g ml−1 (calibration B). The standard working solutions were directly applied to the plasma samples using a calibrated micropipette (Socorex Calibra 10–100 l, Lausanne, Switzerland), followed by a vortex-mixing step (30 s). After 5 min equilibration, the sample preparation procedure was started. The calibration samples were treated in a similar way as the unknown samples. Peak area ratios between levamisole and the IS were plotted against the concentration ratios. The correlation coefficients (r) and the goodness-of-fit coefficients (g) were calculated [9,11]. The within-run precision and trueness were evaluated by analyzing at least six blank plasma samples, which were spiked with levamisole at a high (2.0 g ml−1 ) and low (0.1 g ml−1 ) concentration level on the same day. The trueness (i.e. the closeness of agreement between the true value and the mean result which was obtained by applying the experimental procedure several times) had to fall between −20 and 10% [7,9,10,12]. The within-run precision covered the repeatability, i.e. by using the same method on identical test material, in the same laboratory, by the same operator, using the same equipment within short intervals of time. It was expressed as the relative standard deviation (R.S.D., %) and had to be below the
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R.S.D.max (with R.S.D.max = 2(1−0.5 log conc) × 2/3) [9,10,12]. The results of the quality control (QC) samples demonstrated the between-run precision or reproducibility (i.e. in the same laboratory, but eventually a different operator or apparatus and between analytical batches). The between-run precision had to be below the R.S.D.max (with R.S.D.max = 2(1−0.5 log conc) ). The LOQ was defined as the lowest concentration of levamisole for which the method was validated with a trueness and precision that fall within the recommended ranges (trueness: −20 to 10%, precision: R.S.D. < R.S.D.max with R.S.D.max = 2(1−0.5 log conc) × 2/3) [9]. The LOQ was also established as the lowest point of the calibration graph. The LOQ was determined by analyzing at least six blank plasma samples which were spiked with levamisole at a concentration of 0.025 g ml−1 (LC–MS/MS method) or 0.1 g ml−1 (LC-UV method). The LOD was defined as the lowest concentration of levamisole that could be recognized by the detector with a signal-to-noise ratio (S/N) of ≥3 [12]. The LOD values were calculated, using spiked plasma samples (0.025 or 0.1 g ml−1 in case of the LC–MS/MS or LC-UV methods, respectively). The selectivity of the method was demonstrated by analyzing blank plasma samples from pigs that did not receive any medication (interference of endogenous compounds). Other specificity experiments (interference of analogous compounds) were not performed because levamisole is the only compound belonging to the group of imidazolthiazoles which is used in veterinary medicine [10].
3. Results and discussion 3.1. Isolation of the compounds To extract levamisole (and the IS) from the animal plasma, a liquid–liquid extraction based on the procedure described by Woestenborghs et al. [1], was evaluated during preliminary experiments. A mixture of hexane:isoamyl alcohol (95:5, v/v) was used instead of heptane:isoamyl alcohol. Several extraction possibilities were tried out: A: a single liquid–liquid extraction (LLE) in alkaline medium; B: as A, but the LLE was repeated and the combined organic phases were evaporated under N2 ; C: a liquid–liquid back
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extraction; D: as C, but the extraction steps in alkaline medium, were performed twice. Each time, the recovery of levamisole was evaluated together with the extent of appearance of matrix interferences in the chromatogram obtained by LC-UV. The highest extraction recovery was obtained using option A (i.e. 90.9%, n = 3). Because no matrix interferences were seen at the elution zone of levamisole and the IS, this method was preferred. Compared to the methods of Woestenborghs et al. [1], Loussouarn et al. [2] and Kouassi et al. [3], this resulted in an improvement of sample analysis time without loss of sensitivity. The sample preparation procedure for the LC–MS/ MS method could be reduced further to a protein precipitation step using acetonitrile without loss of sensitivity (see LOQ values, Table 1) or specificity. Hence, up to 80 samples could be processed per day by one analyst. 3.2. Chromatography The chromatographic procedure was based on that described by Cherlet et al. [8] For LC-UV analysis, a gradient elution was performed in order to remove late eluting endogenic compounds from the column. Fig. 2 shows the LC-UV chromatograms of a blank plasma sample (A), a sample spiked at 0.5 g ml−1 for LEV and 2 g ml−1 for the IS (B) and an incurred plasma sample (C, LEV concentration: 1.22 g ml−1 , IS concentration: 2 g ml−1 ). As can be seen, no interferences of endogenic compounds were observed at the elution zone of levamisole and the IS. Some modifications were performed to make the LC-UV method suitable for LC–MS/MS application. First, the flow rate was reduced from 1 to 0.2 ml min−1 . Second, the mobile phase composition was slightly modified (A/B, 60/40%, v/v, instead of 65/35%, v/v), so that elution could be performed isocratically. In addition, the run time was reduced from 20 to 10 min, resulting in a greater sample throughput rate. 3.3. Mass spectrometry The chemical structures, as well as the tandem mass spectra of levamisole and methyllevamisole are shown in Fig. 1. Under the conditions applied, the protonated molecular ions [M−H]+ of levamisole and
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Table 1 Validation results for the determination of levamisole in pig plasma LC-UV Linearity Concentration ranges 0–0.5 0–4.0
LC–MS/MS
r
g (%)
r
g (%)
(g ml−1 ) 0.9955 0.9997
9.7 2.8
0.9998 0.9994
1.7 2.2
Precision and trueness
Mean (g ml−1 )
R.S.D. (%)
Trueness (%)
Mean (g ml−1 )
R.S.D. (%)
Trueness (%)
Within-run 0.1 g ml−1 (n = 6) 2.0 g ml−1 (n = 6)
0.098 1.91
6.8 6.9
−1.9 −4.6
0.096 2.04
1.7 3.6
−4.3 1.9
Between-run 0.1 g ml−1 (n = 6) 2.0 g ml−1 (n = 6)
n.d. n.d.
n.d. n.d.
n.d. n.d.
0.098 2.08
4.6 3.4
−2.2 4.2
LOQ (n = 6)
0.098
6.8
−1.9
0.023
4.4
−8.4
LOD
0.077 g ml−1
0.009 g ml−1
R.S.D.max —within-day: 0.025 g ml−1 , 18.6%; 0.1 g ml−1 , 15.1%; 2.0 g ml−1 , 9.6%. Between day 0.1 g ml−1 , 22.6%; 2.0 g ml−1 , 19.4%; n.d.: not determined.
the IS (m/z = 205.1 and 219.2, respectively) were fragmented for a large part. For each compound, two prominent product ions were formed: at m/z = 145.1 and 178.2 for levamisole and at m/z = 159.2 and 192.2 for the IS. The product ions of both compounds show the same mass differences with respect to their protonated molecular ions, indicating that the fragmentation route is the same. The ions with m/z values of 178.2 (LEV) and 192.2 (IS) correspond probably with the loss of a HCN molecule; those with m/z values of 145.1 (LEV) and 159.2 (IS) with an additional loss of SH• . For quantification, the most abundant product ions at m/z = 178.2 and m/z = 192.2 were used for levamisole and the IS. Fig. 3 shows the chromatograms obtained by LC–MS/MS for a blank sample (A), a plasma sample spiked with levamisole at 0.5 g ml−1 and the IS at 2 g ml−1 (B) and the same incurred plasma sample as in Fig. 2C (LEV concentration: 1.08 g ml−1 , IS concentration: 2 g ml−1 ). As can be seen from Figs. 2A and 3A, the chromatograms are much cleaner than those obtained by LC-UV, due to the higher specificity of LC–MS/MS. In addition, the LC–MS/MS method is more sensitive as is demonstrated by the S/N ratios of the levamisole peaks (concentration 0.5 g ml−1 ) in Figs. 2B and 3B.
3.4. Method validation Linearity: Linearity of response was evaluated for both the LC-UV and LC–MS/MS methods. The calibration graphs were linear over the ranges tested (0–0.5 and 0–4.0 g ml−1 ). Each time, the correlation coefficients (r) and the goodness-of-fit coefficients (g) were determined. The results fell within the ranges specified (r ≥ 0.99, g ≤ 10%) and are shown in Table 1 [9,11]. Precision and trueness: Within-run precision and trueness were investigated for the determination of levamisole at a low (0.1 g ml−1 ) and high (2.0 g ml−1 ) concentration. The results are presented in Table 1. As can be seen, the R.S.D.s were below the maximum R.S.D. values [9,10,12]. The between-run precision was evaluated for the LC–MS/MS method using spiked QC samples (concentration levels 0.1 and 2.0 g ml−1 ) which were analyzed together with each analytical batch of samples. The R.S.D. values fell below the R.S.D.max values of 22.6 and 19.4% for an analyte content of 0.1 and 2.0 g ml−1 , respectively (R.S.D.max = 2(1−0.5 log C) ). The trueness also fell within the accepted range of −20 to 10% of the target (or spiked) values [9,10,12]. Hence, it can be concluded that the criteria for both the
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Fig. 2. Chromatograms obtained by LC-UV of a blank plasma sample (A), a sample spiked at 0.5 g ml−1 (LEV) and 2 g ml−1 (IS) (B) and an incurred sample (C, LEV concentration: 1.22 g ml−1 , IS concentration: 2 g ml−1 ).
precision and trueness are met for the determination of levamisole, according to both methods. LOQ: The LOQ for the determination of levamisole was established at a level of 0.025 g ml−1 for the LC–MS/MS method and 0.1 g ml−1 for the LC-UV method. As can be seen from Table 1, both the precision and trueness fell within the recommended ranges. The LOQ was also established as the lowest point of the calibration graphs. LOD: The LOD values for levamisole were 0.009 and 0.077 g ml−1 for the LC–MS/MS and LC-UV methods, respectively. As can be seen from the results in Table 1, lower LOD and LOQ values were obtained when the LC–MS/MS technique was used. This can be attributed to the higher sensitivity and specificity of the triple quadrupole LC–MS/MS technique, when used in the MRM mode. Hence, the observed LOD value for the LC–MS/MS method is lower than those reported in the literature (Woestenborghs et al. [1]:
0.010 g ml−1 , Vandamme et al. [6]: 0.021 g ml−1 , Alvinerie et al. [4]: 0.050 g ml−1 ). Specificity: The described method proved to be specific for levamisole with respect to the interference of endogenous compounds (cf. Figs. 2A and 3A). 3.5. Analysis of biological samples The results of the analysis of incurred plasma samples are shown in Table 2. A scatter plot and a residual plot is shown in Fig. 4A and 4B, respectively. As can be seen, a good correlation (correlation coefficient, r 2 = 0.9831) is obtained between the results of the LC-UV and LC–MS/MS methods. The intercept and slope (including the 95% confidence interval) were 0.005 g ml−1 (−0.122 to 0.132 g ml−1 ) and 1.13 (1.01 to 1.25), respectively. The standard error (sy/x ) was 0.103 g ml−1 . From these results, it can be concluded that both methods are equally valuable for
Fig. 3. Chromatograms obtained by LC–MS/MS of a blank plasma sample (A), a sample spiked at 0.5 g ml−1 (LEV) and 2 g ml−1 (IS) (B) and an incurred sample (C, LEV concentration: 1.08 g ml−1 , IS concentration: 2 g ml−1 ).
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Fig. 4. Line fit plot (A) and residual plot (B) of the results of the plasma analysis of incurred samples using the LC–MS/MS and LC-UV methods.
application in the field of pharmacokinetic and residue analysis. However, each method has its special features: the LC–MS/MS method is characterized by a high speed of analysis and excellent sensitivity and
specificity. The LC-UV method has the advantage of being more accessible, since not every routine lab has an LC–MS/MS apparatus.
Table 2 Results of the LC-UV and LC–MS/MS analyses of plasma samples from two pigs that received levamisole via drinking water (8 mg kg−1 , 8 h period)
4. Conclusions
LEV concentration (g ml−1 )
Time (h)
LC-UV
0 0.5 1 2 4 7 16 24 40
LC–MS/MS
Pig 1
Pig 2
Pig 1
Pig 2
1.58 1.63 1.50 1.40 1.26 1.05 0.41 0.18
1.45 1.51 1.60 1.19 1.22 0.93 0.25
1.26 1.33 1.30 1.32 1.24 1.01 0.32 0.14
1.24 1.26 1.30 1.22 1.08 0.89 0.19 0.08
This paper has described a LC-UV and a LC–MS/ MS method for the determination of levamisole in animal plasma. Both methods were completely validated according to EU regulations (linearity, precision, trueness, LOQ, LOD and specificity) and good results were obtained. Incurred plasma samples from pigs that received levamisole via drinking water were quantitatively analyzed using the described methods. A good correlation between the results of both methods was obtained, proving their usefulness. However, the LC–MS/MS method was considered superior to our LC-UV method and to other methods in the literature, due to the possibility of a high sample throughput (up to 80
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samples a day) and excellent specificity and sensitivity. [8]
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accordance with Council Regulation (EEC) 2377/90, vol. 6, Brussels, Luxembourg, 1991, pp. 102–111. M. Cherlet, S. De Baere, S. Croubels, P. De Backer, J. Chromatogr. 742 (2000) 283. European Commission, Directorate General for Public Health and Consumers Protection, Draft Commission Decision Laying Down Performance Criteria for the Analytical Methods to be Used for Detecting Certain Substances and Residues Thereof in Live Animal and Animal Products According to Council Directive 96/23/EC repealing Commission Decision 90/515/EC, 93/256/EC and 96/257/EC, SANCO/1805/2000, 18 July 2000. Commission of the European Communities, Notice to Applicants, Veterinary Medicinal Products: Establishment of Maximum Residue Limits (MRLs) for Residues of Veterinary Products in Foodstuffs of Animal Origin, Draft, vol. 8, Development and Validation of a Proposed Regulatory Method, EMEA/CVMP/573/00. J. Knecht, G. Stork, Z. Anal. Chem. 270 (1974) 97. R.J. Heitzman, Veterinary Drug Residues, Report Eur. 14126-EN, Commission of the EC, Brussels, Luxembourg, 1992.
Liquid chromatographic determination of levamisole in animal plasma: ultraviolet versus tandem mass spectrometric detection Siegrid De Baere∗ , Marc Cherlet, Siska Croubels, Kris Baert, Patrick De Backer Departments of Pharmacology, Pharmacy and Toxicology, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, B-9820 Merelbeke, Belgium Received 2 July 2002; received in revised form 1 October 2002; accepted 22 October 2002
Abstract This work presents the optimization and validation of a liquid chromatography (LC)–ultraviolet (UV) detection method and a tandem mass spectrometry (LC–MS/MS) method with positive electrospray ionization for the determination of levamisole, an anthelmintic for veterinary use, in animal plasma. A liquid–liquid extraction procedure in alkaline medium, using hexane-isoamyl alcohol (95:5, v/v) as extraction solvent, was performed to clean-up the plasma samples prior to LC-UV analysis. The sample preparation prior to LC–MS/MS analysis consisted of a protein precipitation step, using acetonitrile. Methyllevamisole was used as the internal standard. Chromatographic separation was achieved on a LiChrospher® 60 RP-select B (5 m) column using a mixture of 0.1 M ammonia acetate in water and acetonitrile as the mobile phase. A gradient (flow rate 1 ml min−1 ) or isocratic (flow rate 0.2 ml min−1 ) elution was performed in case of the LC-UV and LC–MS/MS methods, respectively. UV detection was at 235 nm. The mass spectrometer was operated in the multiple reaction monitoring (MRM) mode. The methods were validated (linearity, precision, trueness, limit of quantification, limit of detection, specificity) according to the requirements defined by the European Community. Good linearity was achieved over the concentration ranges tested (0–0.5 g ml−1 and 0–4.0 g ml−1 , r > 0.99, goodness-of-fit <10%). Limits of quantification of 0.025 g ml−1 and 0.1 g ml−1 were achieved for the LC–MS/MS and LC-UV methods, respectively. The limits of detection were 0.009 and 0.077 g ml−1 , respectively. The results for precision (within-day and between-day) fell within the ranges specified. The methods have been successfully used for the determination of levamisole in plasma samples from two pigs medicated via drinking water. A good correlation was observed between the results of both methods (r 2 = 0.9831, slope = 1.13, intercept = 0.005 g ml−1 ), proving their usefulness for the application in the field of pharmacokinetic and residue analysis. © 2002 Elsevier Science B.V. All rights reserved. Keywords: Levamisole; Anthelmintic; Animal plasma; Liquid chromatography-ultraviolet; Liquid chromatography–mass spectrometry/MS; Tandem mass spectrometry; Validation; Pharmacokinetics; Residue analysis
1. Introduction Levamisole, (S)-(−)-2,3,5,6-tetrahydro-6-phenylimidazo-[2,1b]thiazole (LEV, Fig. 1) is a potent broad-spectrum anthelmintic drug, which is widely ∗ Corresponding author. Tel.: +32-9-264-73-24; fax: +32-9-264-74-97. E-mail address: [email protected] (S. De Baere).
used in veterinary medicine for the control of gastrointestinal parasites in cattle, sheep and pigs. It is normally administered orally, by pour-on or by subcutaneous or intramuscular injection. The recommended dose is 8 mg kg−1 body weight (BW). Various techniques have been used for the determination of levamisole in biological fluids. They include gas chromatography with nitrogen–phosphorus detection (GC-NPD) [1,2] and flame ionization detection
0003-2670/03/$ – see front matter © 2002 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 3 - 2 6 7 0 ( 0 2 ) 0 1 3 7 7 - 6
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Fig. 1. Chemical structures and tandem mass spectra of levamisole and methyllevamisole obtained after direct infusion of standard solutions of 10 g ml−1 of both components.
(GC-FID) [3] and liquid chromatography (LC) with ultraviolet (UV) detection [4–6]. No GC–mass spectrometry (GC–MS) or LC–MS methods have been reported for the determination of levamisole in plasma. For the GC methods, the clean-up of the plasma samples consists of a rather time consuming liquid–liquid back extraction procedure, consecutively in alkaline, acidic and alkaline media [1–3]. Loussouarn et al. also described a solid-phase extraction (SPE) procedure using octyl (C8) cartridges [2]. The sample preparation for the LC methods is generally less complicated and consists of a liquid–liquid extraction procedure in alkaline medium [5,6] or a SPE procedure using octadecyl (C18) columns. Gas chromatography is performed on glass columns packed with 3% OV-17 on Supelcoport [1,3] or Gas-Chrom Q (80–100 mesh) [2]. Reversed-phase ion-pair chromatography is applied for the elution of levamisole from the LC columns. Ultraviolet detection was performed at 225 nm. Some authors mention the use of an internal standard (IS, e.g. methyllevamisole [1]; trimipramine [3]; quinine [6]) in order to ameliorate the quantification of levamisole. The reported limit of quantification (LOQ) and limit of detection (LOD) values are generally lower for the GC than for the LC methods (GC—LOD: 10 ng ml−1 [1,2], LOQ: 1.9 g ml−1 [3]; LC-UV— LOD: 21–50 ng ml−1 [4,6], LOQ: 72–80 ng ml−1 [5,6]).
The purpose of this work was to develop a method for the determination of levamisole in animal plasma which could be used for pharmacokinetic and residue studies with animals. Therefore, a LC-UV method was optimalized in the first instance. Because many samples have to be analyzed in pharmacokinetic studies, special attention has been paid to enhanced speed of analysis, without loss of sensitivity and specificity. Hence, a LC–MS/MS method was developed subsequently. Both methods were fully validated according to the requirements defined by the European Community (linearity, precision and accuracy, LOQ and LOD, specificity) [7]. The practical applicability of the methods was evaluated during the routine analysis of plasma samples which were obtained during a pharmacokinetic and residue study in pigs after the administration of levamisole via drinking water (dose level: 8 mg kg−1 body weight; administration period: 8 h).
2. Experimental 2.1. Standards and chemicals Levamisole hydrochloride was a chemical reference substance (CRS) of the European Pharmacopoeia (Strasbourg, France). A stock solution of 1000 g ml−1 was prepared in methanol. It was
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stored at ≤−15 ◦ C and replaced by a fresh stock solution every 3 months. Working solutions at levamisole concentrations of 100, 80, 60, 40, 20, 10 and 5 g ml−1 (calibration graph A) and 10, 8, 6, 4, 2, 1 and 0.5 g ml−1 (calibration graph B) were prepared by appropriate dilution of the stock solution with water. By spiking 1 ml (LC-UV method) or 500 l (LC–MS/MS method) of plasma with 50 or 25 l of each working solution, respectively, calibration graphs ranging from 0.25 to 4 g ml−1 (calibration graph A) and 0.025 to 0.5 g ml−1 (graph B) were obtained. All the working solutions were stored in the refrigerator (2–8 ◦ C) and were replaced by a fresh solution every month. Methyllevamisole hydrochloride (Fig. 1), used as the (IS) was purchased from Janssen Pharmaceutica (Beerse, Belgium). A stock solution of 1000 g ml−1 methyllevamisole was prepared in methanol. Two working solutions at concentrations of 40 and 4 g ml−1 were prepared by diluting the stock solution with water. Storage conditions were the same as for the levamisole stock and working solutions. All solvents used for the mobile phase (acetonitrile, tetrahydrofuran (THF) and water) were of LC grade and were purchased from Acros (Geel, Belgium). All other products and solvents used for the preparation of stock solutions and for extraction were of analytical reagent grade and were purchased from Merck (Darmstadt, Germany—methanol, hexane, isoamyl alcohol, triethylamine and sodium hydroxide pellets) and from Sigma (Bornem, Belgium—ammonium acetate). 2.2. Biological samples Known levamisole-free plasma samples were obtained from pigs which did not receive any medication. Incurred plasma samples were obtained from two pigs which received levamisole via the drinking water at a dose rate of 8 mg kg−1 body weight during an 8 h period. Blood samples were taken before the start of drug administration and at regular time intervals after the end of the medication period (0, 0.5, 1, 2, 4, 7, 16, 24 and 40 h) in identified heparinized tubes. All samples were centrifuged within 1 h after sampling and the plasma was transferred in identified plastic tubes. The samples were stored at ≤−15 ◦ C pending analysis.
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2.3. Apparatus LC-UV system: The LC-UV system consisted of a ternary gradient pump Model 9012, an autosampler Model 410 ProStar and a DAD detector Model ProStar, all from Varian (Walnut Creek, CA). Detection was performed at 235 nm. Quantification was done using the STAR software (Varian). LC–MS/MS system: The LC system consisted of an Alliance type 2690 Separations and a Column Heater Module, both from Waters (Milford). The LC column effluent was pumped to a Quattro Ultima triple quadrupole mass spectrometer (Micromass, Manchester, UK), equipped with an electrospray ionization (ESI) Z-spray source, which was used in the positive ion mode. Quantification was done with the Masslynx software (Micromass) using the mentioned multiple reaction monitoring (MRM) transitions. 2.4. Sample preparation procedure LC-UV method: One milliliter of plasma was transferred into a Pyrex extraction tube and 50 l of the appropriate (IS) working solution was added (40 or 4 g ml−1 , depending on the expected levamisole concentration in the sample). After vortex mixing for 15 s, 2 ml of 1 M sodium hydroxide and 4 ml of the extraction solvent (hexane/isoamyl alcohol, 95:5, v/v) were added and the samples were extracted by rotation (using a home-made mechanical rotation apparatus) for 10 min, followed by a 10-min centrifugation step (2500 rpm, Jouan type C412, Vel, Leuven, Belgium). Thereafter, the organic phase was transferred into a small pyrex tube and evaporated under a gentle stream of nitrogen gas at 40–45 ◦ C (Pierce Reacti-ThermTM heating module and evaporating unit type 18,780, Vel). The dry residue was reconstituted in 250 l of mobile phase A, vortex mixed for 15 s and transferred into an autosampler vial. A 100 l aliquot was injected into the LC-UV system. LC–MS/MS method: Five hundred microliters of plasma was transferred into an Eppendorf centrifuge tube and 25 l of the appropriate IS working solution (40 or 4 g ml−1 , depending on the concentration expected in the incurred samples) was added. After vortex mixing for 15 s, 500 l of acetonitrile was added, followed by a 15 s vortex-mixing step and a 10 min centrifugation step (10,800 rpm, Abbott centrifuge,
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Abbott Diagnostics Division, Belgium). The supernatant was transferred into an autosampler vial and a 25 l aliquot was injected into the LC–MS/MS system.
For our quantitative purposes, the instrument was operated in the MRM mode, using for each analyte one protonated molecular ion > product ion transition: m/z = 205.1 > m/z = 178.2 for levamisole and m/z = 219.2 > m/z = 192.2 for the IS.
2.5. Chromatography 2.7. Method validation LC-UV system: Chromatographic separation was achieved using a reversed-phase Lichrospher® RP-select B (5 m) column (125 mm × 4 mm i.d.), in combination with a guard column of the same type (4 mm × 4 mm i.d.), from Merck. The mobile phase A was a solution of 0.1 M ammonium acetate in water, containing 7.7% (v/v) of tetrahydrofuran and 0.3% (v/v) of triethylamine, while the mobile phase B was pure acetonitrile. A gradient elution was performed: 0–7 min: 65% A, 35% B; 7 to 8 min: linear gradient from 65% A to 50% A; 8–14 min: 50% A, 50% B; 14 to 15 min: linear gradient from 50% A to 65% A; 15–20 min: 65% A, 35% B [8]. Eluate was delivered to the LC column at 1.0 ml min−1 . LC–MS/MS system: Chromatographic separation was achieved using the same type of column and pre-column as for the LC-UV system. The composition of the mobile phase solvents A and B was also the same as those used for the LC-UV system. Mobile phase was delivered to the LC column at 0.2 ml min−1 . An isocratic elution was performed (0–10 min: 60% A, 40% B). 2.6. Mass spectrometry The instrument was calibrated with a solution of sodium iodide according to the manufacturer’s instructions. Thereafter, tuning was performed for the analytes of interest by direct infusion of a 1 g ml−1 solution at 9 l min−1 using a Hamilton syringe (200 l, Bonadoz, Switzerland) and a Harvard pump 11 apparatus (Holliston, USA). The following tuning parameters were used: capillary: 2.8 kV, cone: 30 V, source temperature: 120 ◦ C, desolvation temperature: 250 ◦ C, cone gas flow: ±30 l h−1 , desolvation gas flow: ±600 l h−1 , resolution (LM1, HM1, LM2, HM2): 15.0, ion energy 1:2.0, ion energy 2:0.5, entrance: 20, exit: 15, multiplier: 650 V, Pirani pressure: ±2.9 × 10−3 mbar, dwell time 0.5 s. The optimal settings for collision energy were different for levamisole and the IS, i.e. 24 and 20 eV, respectively.
The proposed methods for the determination of levamisole in animal plasma were validated by a set of parameters which are in compliance with the recommendations as defined by the European Community and with our own criteria which were based on the literature [7]. The linearity of the methods was evaluated by analyzing calibration samples, which were prepared by spiking blank plasma from pigs that did not receive any medication with levamisole. The addition of 50 l (LC-UV method) or 25 l (LC–MS/MS method) of the above mentioned standard working solutions resulted in analyte concentrations of 0.25, 0.5, 1.0, 2.0, 3.0 and 4.0 g ml−1 (calibration A) and 0.025, 0.05 (only for the LC–MS/MS method) 0.1, 0.2, 0.3, 0.4 and 0.5 g ml−1 (calibration B). The standard working solutions were directly applied to the plasma samples using a calibrated micropipette (Socorex Calibra 10–100 l, Lausanne, Switzerland), followed by a vortex-mixing step (30 s). After 5 min equilibration, the sample preparation procedure was started. The calibration samples were treated in a similar way as the unknown samples. Peak area ratios between levamisole and the IS were plotted against the concentration ratios. The correlation coefficients (r) and the goodness-of-fit coefficients (g) were calculated [9,11]. The within-run precision and trueness were evaluated by analyzing at least six blank plasma samples, which were spiked with levamisole at a high (2.0 g ml−1 ) and low (0.1 g ml−1 ) concentration level on the same day. The trueness (i.e. the closeness of agreement between the true value and the mean result which was obtained by applying the experimental procedure several times) had to fall between −20 and 10% [7,9,10,12]. The within-run precision covered the repeatability, i.e. by using the same method on identical test material, in the same laboratory, by the same operator, using the same equipment within short intervals of time. It was expressed as the relative standard deviation (R.S.D., %) and had to be below the
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R.S.D.max (with R.S.D.max = 2(1−0.5 log conc) × 2/3) [9,10,12]. The results of the quality control (QC) samples demonstrated the between-run precision or reproducibility (i.e. in the same laboratory, but eventually a different operator or apparatus and between analytical batches). The between-run precision had to be below the R.S.D.max (with R.S.D.max = 2(1−0.5 log conc) ). The LOQ was defined as the lowest concentration of levamisole for which the method was validated with a trueness and precision that fall within the recommended ranges (trueness: −20 to 10%, precision: R.S.D. < R.S.D.max with R.S.D.max = 2(1−0.5 log conc) × 2/3) [9]. The LOQ was also established as the lowest point of the calibration graph. The LOQ was determined by analyzing at least six blank plasma samples which were spiked with levamisole at a concentration of 0.025 g ml−1 (LC–MS/MS method) or 0.1 g ml−1 (LC-UV method). The LOD was defined as the lowest concentration of levamisole that could be recognized by the detector with a signal-to-noise ratio (S/N) of ≥3 [12]. The LOD values were calculated, using spiked plasma samples (0.025 or 0.1 g ml−1 in case of the LC–MS/MS or LC-UV methods, respectively). The selectivity of the method was demonstrated by analyzing blank plasma samples from pigs that did not receive any medication (interference of endogenous compounds). Other specificity experiments (interference of analogous compounds) were not performed because levamisole is the only compound belonging to the group of imidazolthiazoles which is used in veterinary medicine [10].
3. Results and discussion 3.1. Isolation of the compounds To extract levamisole (and the IS) from the animal plasma, a liquid–liquid extraction based on the procedure described by Woestenborghs et al. [1], was evaluated during preliminary experiments. A mixture of hexane:isoamyl alcohol (95:5, v/v) was used instead of heptane:isoamyl alcohol. Several extraction possibilities were tried out: A: a single liquid–liquid extraction (LLE) in alkaline medium; B: as A, but the LLE was repeated and the combined organic phases were evaporated under N2 ; C: a liquid–liquid back
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extraction; D: as C, but the extraction steps in alkaline medium, were performed twice. Each time, the recovery of levamisole was evaluated together with the extent of appearance of matrix interferences in the chromatogram obtained by LC-UV. The highest extraction recovery was obtained using option A (i.e. 90.9%, n = 3). Because no matrix interferences were seen at the elution zone of levamisole and the IS, this method was preferred. Compared to the methods of Woestenborghs et al. [1], Loussouarn et al. [2] and Kouassi et al. [3], this resulted in an improvement of sample analysis time without loss of sensitivity. The sample preparation procedure for the LC–MS/ MS method could be reduced further to a protein precipitation step using acetonitrile without loss of sensitivity (see LOQ values, Table 1) or specificity. Hence, up to 80 samples could be processed per day by one analyst. 3.2. Chromatography The chromatographic procedure was based on that described by Cherlet et al. [8] For LC-UV analysis, a gradient elution was performed in order to remove late eluting endogenic compounds from the column. Fig. 2 shows the LC-UV chromatograms of a blank plasma sample (A), a sample spiked at 0.5 g ml−1 for LEV and 2 g ml−1 for the IS (B) and an incurred plasma sample (C, LEV concentration: 1.22 g ml−1 , IS concentration: 2 g ml−1 ). As can be seen, no interferences of endogenic compounds were observed at the elution zone of levamisole and the IS. Some modifications were performed to make the LC-UV method suitable for LC–MS/MS application. First, the flow rate was reduced from 1 to 0.2 ml min−1 . Second, the mobile phase composition was slightly modified (A/B, 60/40%, v/v, instead of 65/35%, v/v), so that elution could be performed isocratically. In addition, the run time was reduced from 20 to 10 min, resulting in a greater sample throughput rate. 3.3. Mass spectrometry The chemical structures, as well as the tandem mass spectra of levamisole and methyllevamisole are shown in Fig. 1. Under the conditions applied, the protonated molecular ions [M−H]+ of levamisole and
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Table 1 Validation results for the determination of levamisole in pig plasma LC-UV Linearity Concentration ranges 0–0.5 0–4.0
LC–MS/MS
r
g (%)
r
g (%)
(g ml−1 ) 0.9955 0.9997
9.7 2.8
0.9998 0.9994
1.7 2.2
Precision and trueness
Mean (g ml−1 )
R.S.D. (%)
Trueness (%)
Mean (g ml−1 )
R.S.D. (%)
Trueness (%)
Within-run 0.1 g ml−1 (n = 6) 2.0 g ml−1 (n = 6)
0.098 1.91
6.8 6.9
−1.9 −4.6
0.096 2.04
1.7 3.6
−4.3 1.9
Between-run 0.1 g ml−1 (n = 6) 2.0 g ml−1 (n = 6)
n.d. n.d.
n.d. n.d.
n.d. n.d.
0.098 2.08
4.6 3.4
−2.2 4.2
LOQ (n = 6)
0.098
6.8
−1.9
0.023
4.4
−8.4
LOD
0.077 g ml−1
0.009 g ml−1
R.S.D.max —within-day: 0.025 g ml−1 , 18.6%; 0.1 g ml−1 , 15.1%; 2.0 g ml−1 , 9.6%. Between day 0.1 g ml−1 , 22.6%; 2.0 g ml−1 , 19.4%; n.d.: not determined.
the IS (m/z = 205.1 and 219.2, respectively) were fragmented for a large part. For each compound, two prominent product ions were formed: at m/z = 145.1 and 178.2 for levamisole and at m/z = 159.2 and 192.2 for the IS. The product ions of both compounds show the same mass differences with respect to their protonated molecular ions, indicating that the fragmentation route is the same. The ions with m/z values of 178.2 (LEV) and 192.2 (IS) correspond probably with the loss of a HCN molecule; those with m/z values of 145.1 (LEV) and 159.2 (IS) with an additional loss of SH• . For quantification, the most abundant product ions at m/z = 178.2 and m/z = 192.2 were used for levamisole and the IS. Fig. 3 shows the chromatograms obtained by LC–MS/MS for a blank sample (A), a plasma sample spiked with levamisole at 0.5 g ml−1 and the IS at 2 g ml−1 (B) and the same incurred plasma sample as in Fig. 2C (LEV concentration: 1.08 g ml−1 , IS concentration: 2 g ml−1 ). As can be seen from Figs. 2A and 3A, the chromatograms are much cleaner than those obtained by LC-UV, due to the higher specificity of LC–MS/MS. In addition, the LC–MS/MS method is more sensitive as is demonstrated by the S/N ratios of the levamisole peaks (concentration 0.5 g ml−1 ) in Figs. 2B and 3B.
3.4. Method validation Linearity: Linearity of response was evaluated for both the LC-UV and LC–MS/MS methods. The calibration graphs were linear over the ranges tested (0–0.5 and 0–4.0 g ml−1 ). Each time, the correlation coefficients (r) and the goodness-of-fit coefficients (g) were determined. The results fell within the ranges specified (r ≥ 0.99, g ≤ 10%) and are shown in Table 1 [9,11]. Precision and trueness: Within-run precision and trueness were investigated for the determination of levamisole at a low (0.1 g ml−1 ) and high (2.0 g ml−1 ) concentration. The results are presented in Table 1. As can be seen, the R.S.D.s were below the maximum R.S.D. values [9,10,12]. The between-run precision was evaluated for the LC–MS/MS method using spiked QC samples (concentration levels 0.1 and 2.0 g ml−1 ) which were analyzed together with each analytical batch of samples. The R.S.D. values fell below the R.S.D.max values of 22.6 and 19.4% for an analyte content of 0.1 and 2.0 g ml−1 , respectively (R.S.D.max = 2(1−0.5 log C) ). The trueness also fell within the accepted range of −20 to 10% of the target (or spiked) values [9,10,12]. Hence, it can be concluded that the criteria for both the
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Fig. 2. Chromatograms obtained by LC-UV of a blank plasma sample (A), a sample spiked at 0.5 g ml−1 (LEV) and 2 g ml−1 (IS) (B) and an incurred sample (C, LEV concentration: 1.22 g ml−1 , IS concentration: 2 g ml−1 ).
precision and trueness are met for the determination of levamisole, according to both methods. LOQ: The LOQ for the determination of levamisole was established at a level of 0.025 g ml−1 for the LC–MS/MS method and 0.1 g ml−1 for the LC-UV method. As can be seen from Table 1, both the precision and trueness fell within the recommended ranges. The LOQ was also established as the lowest point of the calibration graphs. LOD: The LOD values for levamisole were 0.009 and 0.077 g ml−1 for the LC–MS/MS and LC-UV methods, respectively. As can be seen from the results in Table 1, lower LOD and LOQ values were obtained when the LC–MS/MS technique was used. This can be attributed to the higher sensitivity and specificity of the triple quadrupole LC–MS/MS technique, when used in the MRM mode. Hence, the observed LOD value for the LC–MS/MS method is lower than those reported in the literature (Woestenborghs et al. [1]:
0.010 g ml−1 , Vandamme et al. [6]: 0.021 g ml−1 , Alvinerie et al. [4]: 0.050 g ml−1 ). Specificity: The described method proved to be specific for levamisole with respect to the interference of endogenous compounds (cf. Figs. 2A and 3A). 3.5. Analysis of biological samples The results of the analysis of incurred plasma samples are shown in Table 2. A scatter plot and a residual plot is shown in Fig. 4A and 4B, respectively. As can be seen, a good correlation (correlation coefficient, r 2 = 0.9831) is obtained between the results of the LC-UV and LC–MS/MS methods. The intercept and slope (including the 95% confidence interval) were 0.005 g ml−1 (−0.122 to 0.132 g ml−1 ) and 1.13 (1.01 to 1.25), respectively. The standard error (sy/x ) was 0.103 g ml−1 . From these results, it can be concluded that both methods are equally valuable for
Fig. 3. Chromatograms obtained by LC–MS/MS of a blank plasma sample (A), a sample spiked at 0.5 g ml−1 (LEV) and 2 g ml−1 (IS) (B) and an incurred sample (C, LEV concentration: 1.08 g ml−1 , IS concentration: 2 g ml−1 ).
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Fig. 4. Line fit plot (A) and residual plot (B) of the results of the plasma analysis of incurred samples using the LC–MS/MS and LC-UV methods.
application in the field of pharmacokinetic and residue analysis. However, each method has its special features: the LC–MS/MS method is characterized by a high speed of analysis and excellent sensitivity and
specificity. The LC-UV method has the advantage of being more accessible, since not every routine lab has an LC–MS/MS apparatus.
Table 2 Results of the LC-UV and LC–MS/MS analyses of plasma samples from two pigs that received levamisole via drinking water (8 mg kg−1 , 8 h period)
4. Conclusions
LEV concentration (g ml−1 )
Time (h)
LC-UV
0 0.5 1 2 4 7 16 24 40
LC–MS/MS
Pig 1
Pig 2
Pig 1
Pig 2
1.58 1.63 1.50 1.40 1.26 1.05 0.41 0.18
1.45 1.51 1.60 1.19 1.22 0.93 0.25
1.26 1.33 1.30 1.32 1.24 1.01 0.32 0.14
1.24 1.26 1.30 1.22 1.08 0.89 0.19 0.08
This paper has described a LC-UV and a LC–MS/ MS method for the determination of levamisole in animal plasma. Both methods were completely validated according to EU regulations (linearity, precision, trueness, LOQ, LOD and specificity) and good results were obtained. Incurred plasma samples from pigs that received levamisole via drinking water were quantitatively analyzed using the described methods. A good correlation between the results of both methods was obtained, proving their usefulness. However, the LC–MS/MS method was considered superior to our LC-UV method and to other methods in the literature, due to the possibility of a high sample throughput (up to 80
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samples a day) and excellent specificity and sensitivity. [8]
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