Journal of Pharmaceutical and Biomedical Analysis 89 (2014) 203–212
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Journal of Pharmaceutical and Biomedical Analysis journal homepage: www.elsevier.com/locate/jpba
Simultaneous analysis of antibiotics in biological samples by ultra high performance liquid chromatography–tandem mass spectrometry Rocío Cazorla-Reyes 1 , Roberto Romero-González 1 , Antonia Garrido Frenich 1,∗ , Manuel Angel Rodríguez Maresca 2 , José Luis Martínez Vidal 1 1 2
Research Group “Analytical Chemistry of Contaminants”, Department of Chemistry and Physics, University of Almería, E-04120 Almería, Spain Torrecardenas Hospital, Almería, E-04009 Almería, Spain
a r t i c l e
i n f o
Article history: Received 17 May 2013 Received in revised form 31 October 2013 Accepted 1 November 2013 Available online 15 November 2013 Keywords: Antibiotics Human biological samples Simple extraction Ultra-high performance liquid chromatography Tandem mass spectrometry
a b s t r a c t A rapid and reliable multiclass method was developed for the simultaneous analysis of 21 antibiotics (beta-lactams, aminoglycosides, penicillins, cephalosporins, carbapenems or quinolones) in urine, serum, cerebrospinal fluid (CSF) and bronchial aspirations by ultra-high performance liquid chromatography coupled to tandem mass spectrometry (UHPLC–MS/MS). Prior to chromatographic determination, the analytes were extracted from human biological fluids by simple sample treatments, which imply dilution, liquefaction, or protein precipitation. Several chromatographic conditions were optimized in order to obtain a fast separation ( 324.4 (15) 586.7 > 163.2 (30) 725.6 > 144.2 (15) 300.3 > 142.0 (25) 481.4 > 396.3 (15) 366.2 > 349.4 (10) 586.6 > 569.8 (20) 301.4 > 168.2 (12) 198.1 > 136.2 (5) 406.4 > 317.4 (20) 547.3 > 468.5 (10) 232.1 > 140.2 (12) 362.3 > 318.6 (20) 555.5 > 396.4 (10) 350.2 > 106.1 (15) 402.5 > 384.6 (20) 338.2 > 296.5 (20) 941.2 > 316.5 (15) 540.4 > 398.5 (20) 749.1 > 158.2 (30) 811.2 > 159.2 (40)
468.7 > 145.2 (20) 586.7 > 264.4 (25) 725.6 > 100.1 (20) 300.3 > 126.2 (20) 481.4 > 324.3 (17) 366.2 > 114.1 (20) 586.6 > 513.6 (22) 301.4 > 92.1 (25) 198.1 > 108.1 (10) 406.4 > 276.3 (20) 547.3 > 167.2 (25) 232.1 > 188.3 (10) 362.3 > 221.3 (35) 555.5 > 167.2 (25) 350.2 > 114.1 (30) 402.5 > 364.6 (25) 338.2 > 235.4 (20) 941.2 > 204.2 (20) 540.4 > 182.1 (25) 749.1 > 590.7 (20) 811.2 > 341.6 (25)
a
Collision energies (eV) are given in brackets.
(a) Urine: 0.5 mL of urine samples was diluted with 1 mL of mobile phase (1:2, v/v). The mixture was vigorously shaken prior to UHPLC–MS/MS analysis. (b) Serum: 1 mL of serum was mixed with 1 mL of acetronitrile. After vigorous shaking, samples were centrifuged at 5000 rpm (4136 × g) for 5 min, and then, 250 L of supernatant was taken and diluted with 750 L of mobile phase (1:3, v/v) before chromatographic analysis. (c) CSF: For the treatment of CSF samples, 800 L of mobile phase was added to 200 L of CSF (1:4, v/v) and they were vigorously shaken. After that, they were injected directly into chromatographic system. (d) Bronchial aspirations: 4 mL of an aqueous solution of DTT (1%, w/v) was added for each gram of sample. After that, samples were vigorously shaken and they were centrifuged at 5000 rpm (4136 × g) for 5 min. Then 1 mL of supernatant was taken and injected directly into UHPLC–MS/MS. 3. Results and discussion 3.1. MS/MS analysis The optimization of MS/MS parameters (cone voltage and collision energy) was performed by infusion of a standard solution of 20 mg/L of each antibiotic in a mixture of methanol:water (50:50, v/v) and 50 L of formic acid (except for sulbactam an clavulanic acid) at a flow rate of 10 L/min. The compounds were injected into the ESI source in positive ion mode at different voltages, except for sulbactam and clavulanic acid, which were monitored in negative ion mode ([M−H]− ). Under the experimental conditions, protonated molecules ([M+H]+ ) were obtained in all the cases, except for meropenem and piperacillin, which formed sodium adduct (m/z 406.4 and m/z 540.4, respectively). For vancomycin, despite molecular weight was 1449.2, the monitored ion was m/z 725.6, because this compound has multiple basic ionizable groups and the diprotonated molecule, [M+2H]2+ , provided the most sensitive signal. The same situation was observed for daptomycin, monitoring the ion at m/z 811.2, despite the molecular weight was 1620.7 [39,40]. Then, collision energies were studied in order to find the most abundant product ions, selecting the most sensitive transition for quantification purposes and a second one for confirmation.
Table 1 shows MS/MS transitions as well as cone voltages and collision energy values optimized for the selected compounds. Although non-specific transitions were avoided, for some compounds such as tigecycline, amoxicillin and moxifloxacin, the most sensitive detected product ions correspond with the loss of ammonia [M+H−NH3 ]+ . 3.2. Chromatographic separation Chromatographic conditions were studied in order to achieve the best separation and retention for the analytes, minimizing analysis time. First, several experiments were performed testing different mobile phases consisting of methanol as organic phase and water with ammonium formate (5 mM) or different concentrations of formic acid (0.01, 0.05 and 0.1%, v/v). The addition of formic acid provided better results than ammonium formate, and it improved the ionization efficiency. Furthermore, 0.01% (v/v) of formic acid provided better peak shape, and therefore methanol, as organic modifier, and an aqueous solution of formic acid 0.01%, v/v were used as mobile phase. Several gradient profiles were studied, and good response was obtained with the gradient described in Section 2.2. Other parameters such as column temperature, flow rate and injection volume were studied in order to get a fast and reliable separation, and the best results were obtained when 30 ◦ C was used as column temperature, 0.3 mL/min as flow rate and 5 L were injected. Under these conditions, retention time of the antibiotics was constant, ranging from 0.61 min (tobramycin) to 3.82 min (daptomycine). The analytes were distributed in seven overlapping acquisition functions, using a maximum of 4 antibiotics (8 transitions) per function. Because there are several compounds detected in positive ion mode that co-eluted with clavulanic acid, which was detected in negative ion mode, polarity switching was not an option and therefore, two injections were carried out, one to monitor the compounds detected in positive ion mode (see Table 1) and the second one to monitor clavulanic acid and sulbactam. Finally, it must be highlighted that one the main advantages of UHPLC is that it is able to perform a run in 6.0 min (positive mode) and 5.8 min (negative mode), minimizing the running time necessary for the detection of the selected compounds, and therefore, it
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Fig. 1. UHPLC–MS/MS chromatograms obtained from a blank serum sample spiked at 1 mg/L.
can be used in routine analysis. Fig. 1 shows a representative chromatogram obtained from a spiked standard mixture at 1 mg/L of the selected antibiotics in serum. 3.3. Optimization of the extraction procedure Extraction procedure is the critical step, when a multiclass antibiotic method has to be developed, due to the different physicochemical properties of the compounds that have to be extracted simultaneously. Generally, antibiotic extraction from biological fluids includes laborious stages of evaporation, elution or cleanup [36]. However, single extraction procedures should be developed for the simultaneous extraction of antibiotics, which cover a wide range of polarity in urine, serum, CSF and bronchial aspirations, in order to reduce sample handling and increase sample throughput. For urine matrix, a simple dilution of sample was investigated. For that, several volumes of spiked urine (2 mg/L) were diluted with mobile phase (1:1 (v/v) of an aqueous solution of formic acid 0.01% (v/v) and methanol). Dilutions 1:1, 1:2, 1:4, 1:9 and 1:100 (v/v) (sample-mobile phase) were tested. It was noted that the more diluted the sample was, less matrix effect was observed; however, the sensitivity was lower. In this sense, better results were obtained when a dilution factor of 1:2 was used, taking into account that this dilution factor provided good sensitivity and minimized the presence of interferences, as well as suitable peak shape was obtained. In the case of serum, 1 mL of acetonitrile was added to 1 mL of spiked sample (2 mg/L) in order to precipitate the proteins present in the matrix. After that, a centrifugation step was carried out. It was observed that 5000 rpm (4136 × g) should be used to get a clean supernatant layer. In order to reduce the burden of matrix
and eliminate the presence of interferences, several dilutions with mobile phase, were evaluated, including 1:1, 1:3 and 1:9 (v/v). Better results were obtained when a dilution factor of 1:3 (v/v) was employed in terms of sensitivity and matrix effect. Finally, a SPE procedure was evaluated in order to improve the sensitivity, bearing in mind that target analytes could be concentrated during the extraction stage. For that purpose, two types of cartridges, Oasis HLB and C18, were evaluated, and they were conditioned with 5 mL of methanol and 5 mL of water. After that, the diluted supernatant was passed through the cartridges. They were dried during 30 min and target compounds were eluted using a mixture of methanol:water (1:1, v/v). When this procedure was applied, recoveries did not improve, and therefore, it was not applied in further experiments. For CSF, spiked samples (2 mg/L) were diluted with mobile phase (1:1, 1:4, 1:9 (v/v)). The best recoveries were obtained when samples were diluted 1:4 (v/v). Moreover, suitable response was achieved when this dilution was used. Different volumes (0, 50 and 100 L) of a solution of phosphate buffer 0.1 M at pH 7, which is a system for regulation of intra and extra cellular pH [41], were added to CSF samples in order to homogenize the tissue, but recoveries were not improved when these experiments were performed, and therefore, it was not used for further experiments. Finally, the extraction method of antibiotics from bronchial aspirations was optimized. First, 4 mL of an aqueous solution of DTT (1%, w/v) was added to 1 g of spiked sample (2 mg/kg) to liquefy the sample [42]. Then, a centrifugation step at 5000 rpm (4136 × g) for 5 min was carried out and a clean extract was obtained. After that, 500 L of the obtained supernatant was diluted with 500 L of mobile phase (1:1, v/v) and injected into the UHPLC system, and
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Fig. 2. Slope ratios between matrix-matched and solvent calibration of the target antibiotics. Compliance interval covering the range between 0.8 and 1.2 for tolerable matrix effect has been plotted in: (a) urine; (b) serum; (c) CSF and (d) bronchial aspiration. Compounds code: (1) tobramycin; (2) amikacin; (3) vancomycin; (4) imipenem; (5) cefepime; (6) amoxicillin; (7) tigecycline; (8) tazobactam; (9) clavulanic acid; (10) meropenem; (11) ceftazidime; (12) sulbactam; (13) levofloxacin; (14) ceftriaxone; (15) ampicillin; (16) moxifloxacin; (17) linezolid; (18) teicoplanin; (19) piperacillin; (20) clarithromycin; (21) daptomycin.
the results were compared with those obtained injecting the supernatant directly. Bearing in mind that the dilution did not improve the results and worst sensitivity was obtained, the supernatant was injected directly into the UHPLC–MS/MS system. 3.4. Evaluation of matrix effect When ESI is used as ionization technique, one of the main problems is the signal suppression or enhancement of the analytes due to the other components present in the matrix. Moreover, biological fluids are very complex matrices, which have a large amount of compounds that can interfere with the target compounds. Therefore, matrix effect was evaluated in order to ensure bias-free analytical results. For that purpose, five concentration levels (from 0.5 to 5 mg/L) were analyzed in solvent (mixture of methanol:water 1:1, v/v) and in extracted blank samples (matrix). Fig. 2 shows slope ratios matrix/solvent for each compound, and a tolerable signal suppression or enhancement effect was considered if the slope ratio ranged from 0.8 to 1.2, whereas lower values than 0.8 or higher than 1.2 implies a strong effect, signal suppression or enhancement respectively. In the case of urine, it was observed that despite the dilution of the sample, there was a strong matrix effect for most of the compounds except for meropenem, sulbactam, linezolid, teicoplanin, clarithromycin and daptomycine. For these compounds, slope ratio was between 0.8 and 1.2. It can be noted that some compounds (tobramycin, amikacin, vancomycin, imipenem, cefepime, amoxicillin, tigecycline, tazobactam, clavulanic acid, ceftazidime, levofloxacin, ampicillin and moxifloxacin) show a matrix suppression effect (slope ratio < 0.8), whereas a signal enhancement was observed for piperacillin and ceftriaxone (slope ratio > 1.2). When serum was evaluated, matrix effect is not observed for levofloxacin, linezolid, teicoplanin and piperacillin. Furthermore, there was a signal enhancement for meropenem, clarithromycin and daptomycine. The rest of the compounds show a matrix suppression effect. For CSF, amoxicillin, meropenem, sulbactam, levofloxacin, ampicillin, moxifloxacin, linezolid and teicoplanin do not present a matrix effect, observing a matrix enhancement effect for cefepime, piperacillin, clarithromycin and daptomycine. For the rest of the compounds, a matrix suppression effect was observed.
Finally, for bronchial aspiration samples, matrix effect is present in all compounds except for ceftazidime, ceftriaxone and ampicillin. It can be observed a matrix enhancement effect for vancomycin, imipenem, cefepime, amoxicillin, tigecycline, tazobactam, piperacillin and clarithromycin, whereas for the other compounds, a matrix suppression effect is exhibited. Because the existence of matrix effect, despite the dilutions performed in the four matrices evaluated, matrix matched calibration was used for quantification purposes during the determination of the target compounds in samples. 3.5. Validation Performance characteristics of the optimized methods were established by a validation procedure with blank urine, serum, CSF and bronchial aspiration samples, studying selectivity, linearity, limits of detection (LODs) and quantification (LOQs), trueness, repeatability and interday precision. First, a stability study of the compounds in the assayed matrices was carried out spiking blank matrices at 1 mg/L (1 mg/kg in case of bronchial aspiration matrix). The samples were stored at −80 ◦ C and −30 ◦ C for 20 days, by measuring the bias compared to the theoretical concentration (maximum bias accepted ±20%), and the samples were analyzed weekly. It was observed that the compounds were stable for one week if they are stored at −80 ◦ C, whereas at −30 ◦ C, the bias was higher than 20% for some compounds, such as tigecycline, meropenem, clavulanic acid, amoxicillin and ceftriaxone. Bearing in mind that in routine analysis, the results should be provided within 24 h, the samples were stored at −30 ◦ C before analysis. Besides, a short-term stability was also performed, observing that it was guaranteed up to 8 h, if the samples were stored at 8 ◦ C in the autosampler. Method linearity was assayed by means of matrix-matched standard calibration, preparing one calibration curve for every matrix, spiking blank samples with the selected antibiotics in the range from 0.1 to 5 mg/L (from 0.1 to 5 mg/kg for bronchial aspiration matrix). Calibration graphs were obtained by least-squares linear regression analysis, using peak area as analytical response. The responses were linear in the assayed range and determination coefficient (R2 ) values were higher than 0.970, 0.988, 0.970 and 0.990, for urine, serum, CSF and bronchial aspirations, respectively.
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Table 2 Validation parameters of the optimized UHPLC–MS/MS method in urine. Antibiotic
LOD (mg/L)
LOQ (mg/L)
Tobramycin Amikacin Vancomycin Imipenem Cefepime Amoxicillin Tigecycline Tazobactam Clavulanic acid Meropenem Ceftazidime Sulbactam Levofloxacin Ceftriaxone Ampicillin Moxifloxacin Linezolid Teicoplanin Piperacillin Clarithromycin Daptomycin
0.30 0.30 0.30 0.25 0.10 0.10 0.30 0.30 0.10 0.10 0.25 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.25
1.00 1.00 1.00 0.50 0.50 0.50 1.00 1.00 0.50 0.50 0.50 0.50 0.50 0.50 0.50 0.50 0.50 0.50 0.50 0.50 0.50
Recoverya 0.5 mg/L
a b c
–c – – 75 (7) 75 (24) 110 (15) – – 76 (16) 75 (19) 76 (18) 91 (17) 79 (9) 115 (7) 70 (10) 88 (14) 110 (13) 77 (21) 107 (7) 116 (6) 79 (13)
Interday precisionb 1 mg/L
2 mg/L
70 (20) 71 (19) 74 (17) 75 (8) 84 (7) 72 (10) 72 (10) 74 (12) 90 (6) 97 (16) 92 (10) 82 (5) 79 (7) 89 (3) 71 (8) 88 (3) 79 (11) 78 (14) 98 (3) 74 (5) 86 (10)
71 (14) 72 (16) 71 (15) 80 (5) 77 (7) 71 (6) 70 (5) 70 (10) 89 (5) 85 (13) 84 (4) 88 (2) 71 (7) 73 (3) 75 (10) 71 (2) 70 (7) 72 (11) 77 (2) 71 (3) 78 (9)
10 22 21 8 13 18 16 13 7 24 14 10 11 13 16 11 15 18 20 6 7
RSD values of repeatability are given in brackets (n = 5). RSD values obtained at 1 mg/L (n = 5). Not quantified at this concentration.
Deviation of the residuals of each calibration point was also studied, checking it was always equal or lower than 20%. For those samples with concentrations higher than the upper linear concentration range, a dilution with mobile phase was carried out to get a final concentration within the linear range of the method. The sample and the blank sample used for calibration purposes were submitted to this elution step. LODs and LOQs were calculated by analyzing blank samples spiked at 0.01, 0.05, 0.1, 0.2, 0.5, and 1 mg/L (at 0.01, 0.05, 0.1, 0.2, 0.5 and 1 mg/kg for bronchial aspiration samples) and they were determined as the lowest concentration of analyte for which signalto-noise ratios were 3 and 10, respectively (Tables 2–5). LODs were always equal or lower than 0.3 mg/L, whereas LOQs ranged from
0.50 to 1.00 mg/L, from 0.04 to 1.00 mg/L, from 0.01 to 1.00 mg/L and from 0.01 to 0.66 mg/kg in urine, serum, CSF and bronchial aspirations, respectively. It can be observed that lower limits were obtained in CSF, which can be considered a “clean matrix”, and in bronchial aspirations, indicating that the use of DTT is effective for the determination of antibiotics in this type of matrices. Furthermore it can be highlighted that bearing in mind therapeutic doses, the sensitivity of the proposed method was enough to detect the presence of the selected compounds in the assayed matrices. In order to evaluate the trueness of the proposed method, recovery studies were carried out by fortifying blank samples of each matrix with antibiotics at three concentration levels (0.5, 1 and 2 mg/L (0.5, 1 and 2 mg/kg in case of bronchial aspirations)),
Table 3 Validation parameters of the optimized UHPLC–MS/MS method in serum. Antibiotic
LOD (mg/L)
LOQ (mg/L)
Tobramycin Amikacin Vancomycin Imipenem Cefepime Amoxicillin Tigecycline Tazobactam Clavulanic acid Meropenem Ceftazidime Sulbactam Levofloxacin Ceftriaxone Ampicillin Moxifloxacin Linezolid Teicoplanin Piperacillin Clarithromycin Daptomycin
0.04 0.04 0.10 0.40 0.40 0.40 0.30 0.30 0.10 0.20 0.04 0.10 0.04 0.50 0.04 0.04 0.10 0.02 0.04 0.01 0.20
0.10 0.15 0.40 0.20 0.20 0.20 1.00 1.00 0.20 0.50 0.10 0.20 0.10 1.00 0.10 0.10 0.25 0.10 0.15 0.04 0.50
Recoverya 0.5 mg/L
a b c
RSD values of repeatability are given in brackets (n = 5). RSD values obtained at 1 mg/L (n = 5). Not quantified at this concentration.
111 (8) 108 (9) 104 (16) 95 (2) 102 (17) 77 (23) –c – 72 (23) 109 (18) 105 (24) 89 (5) 84 (4) – 77 (13) 95 (3) 76 (8) 77 (22) 74 (18) 70 (11) 91 (24)
Interday precisionb 1 mg/L 74 (11) 86 (5) 100 (10) 83 (5) 71 (15) 81 (12) 70 (20) 71 (20) 73 (4) 84 (10) 90 (14) 95 (4) 76 (2) 75 (20) 87 (6) 80 (2) 80 (4) 78 (13) 73 (14) 76 (5) 101 (20)
2 mg/L 78 (6) 77 (1) 72 (8) 86 (4) 74 (8) 91 (8) 71 (19) 70 (15) 78 (1) 75 (2) 106 (13) 89 (1) 75 (2) 78 (15) 81 (2) 74 (4) 90 (5) 117 (12) 76 (7) 75 (3) 72 (12)
11 5 16 5 20 14 22 20 7 6 14 4 2 23 6 2 4 20 14 8 13
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Table 4 Validation parameters of the optimized UHPLC–MS/MS method in cerebrospinal fluid (CSF). Antibiotic
LOD (mg/L)
LOQ (mg/L)
Tobramycin Amikacin Vancomycin Imipenem Cefepime Amoxicillin Tigecycline Tazobactam Clavulanic acid Meropenem Ceftazidime Sulbactam Levofloxacin Ceftriaxone Ampicillin Moxifloxacin Linezolid Teicoplanin Piperacillin Clarithromycin Daptomycin
0.020 0.300 0.100 0.010 0.142 0.007 0.087 0.300 0.038 0.200 0.001 0.003 0.016 0.064 0.148 0.001 0.002 0.300 0.001 0.001 0.028
0.067 1.000 0.300 0.035 0.474 0.024 0.291 1.000 0.128 0.500 0.003 0.010 0.053 0.215 0.049 0.003 0.007 1.000 0.019 0.001 0.094
Recoverya 0.5 mg/L
a b c
104 (21) –c 70 (20) 90 (19) 110 (13) 93 (3) 89 (21) – 76 (17) 70 (24) 106 (6) 101 (7) 75 (9) 72 (21) 103 (19) 112 (10) 102 (2) – 90 (10) 108 (4) 107 (3)
Interday precisionb 1 mg/L 104 (17) 73 (24) 71 (13) 102 (13) 95 (9) 97 (7) 75 (14) 71 (24) 79 (14) 78 (22) 116 (8) 111 (6) 88 (4) 73 (15) 106 (7) 113 (2) 112 (3) 70 (20) 84 (12) 113 (2) 104 (4)
2 mg/L 1012 (15) 70 (14) 72 (10) 108 (13) 109 (8) 98 (8) 70 (11) 76 (19) 99 (3) 74 (15) 115 (10) 101 (3) 107 (4) 71 (13) 106 (9) 103 (2) 111 (3) 97 (18) 77 (11) 109 (2) 98 (4)
14 19 13 12 24 11 11 19 24 20 14 7 5 14 9 5 6 21 13 5 5
RSD values of repeatability are given in brackets (n = 5). RSD values obtained at 1 mg/L (n = 5). Not quantified at this concentration.
performing 5 replicates at each level (Tables 2–5), observing that adequate recoveries (values ranging from 70 to 120%) were obtained for all the compounds in the four matrices evaluated. Thus, for urine, the obtained values ranged from 70% (ampicillin) to 116% (clarithromycin) for 0.5 mg/L, 70% (tobramycin) to 98% (piperacillin) for 1 mg/L and 70% (tigecycline and tazobactam) to 89% (clavulanic acid) for 2 mg/L. Tobramycin, amikacin, vancomycin, tygecillin and tazobactam were not quantified at 0.5 mg/L in urine matrix. For serum matrix, at 0.5 mg/L, the recoveries ranged from 70% (clarithromycin) to 111% (tobramycin), at 1 mg/L recovery values ranged from 70% (tigecycline) to 101% (daptomycin) and from 70%
(tazobactam) to 117% (teicoplanin) when the samples were spiked with 2 mg/L. In this case, tigecycline, tazobactam and ceftriaxone could not be quantified at 0.5 mg/L. In the case of CSF, the obtained results showed that for 0.5 mg/L, the recoveries ranged from 70% (meropenem) to 112% (moxifloxacin), for 1 mg/L recovery values ranged from 70% (teicoplanin) to 116% (ceftazidime) and from 70% (tigecycline and amikacin) to 115% (ceftazidime) when the samples were spiked with 2 mg/L. The analytes amikacin, tazobactam and teicoplanin could not be quantified at 0.5 mg/L. For bronchial aspirations, the obtained recoveries ranged from 70 (teicoplanin and tazobactam) to 107% (amoxicillin and
Table 5 Validation parameters of the optimized UHPLC–MS/MS method in bronchial aspiration. Antibiotic
LOD (mg/kg)
LOQ (mg/kg)
Tobramycin Amikacin Vancomycin Imipenem Cefepime Amoxicillin Tigecycline Tazobactam Clavulanic acid Meropenem Ceftazidime Sulbactam Levofloxacin Ceftriaxone Ampicillin Moxifloxacin Linezolid Teicoplanin Piperacillin Clarithromycin Daptomycin
0.013 0.027 0.200 0.001 0.002 0.001 0.200 0.002 0.005 0.200 0.200 0.006 0.008 0.196 0.020 0.001 0.200 0.040 0.013 0.005 0.036
0.045 0.090 0.500 0.004 0.006 0.003 0.167 0.008 0.018 0.500 0.667 0.006 0.027 0.655 0.068 0.002 0.667 0.133 0.043 0.016 0.120
Recoverya 0.5 mg/kg
a b c
RSD values of repeatability are given in brackets (n = 5). RSD values obtained at 1 mg/kg (n = 5). Not quantified at this concentration.
94 (13) 102 (20) 87 (18) 106 (15) 100 (18) 107 (11) 111 (17) 70 (20) 71 (23) 97 (23) 106 (18) 105 (3) 107 (16) –c 105 (20) 104 (13) – 70 (20) 105 (17) 106 (12) 105 (7)
Interday precisionb 1 mg/kg
2 mg/kg
90 (3) 109 (16) 82 (14) 102 (3) 91 (9) 119 (4) 79 (12) 74 (15) 72 (13) 84 (17) 94 (10) 81 (2) 101 (6) 107 (12) 102 (7) 100 (1) 78 (3) 77 (14) 110 (5) 112 (3) 104 (4)
99 (1) 72 (10) 87 (13) 76 (3) 73 (9) 78 (4) 72 (9) 79 (11) 71 (14) 70 (12) 101 (8) 79 (3) 73 (9) 77 (10) 76 (13) 75 (1) 90 (6) 95 (12) 74 (5) 77 (2) 80 (8)
5 21 17 5 17 8 12 13 18 18 13 4 10 19 9 4 5 16 10 9 7
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Table 6 Concentration of antibiotics found in real samples from ten patients.a Patient
Antibiotic
Urine
Serum
CSF
Bronchial aspirations
P1
Amoxicillin Clavulanic acid
122.8 22.5
0.7 1.0
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Journal of Pharmaceutical and Biomedical Analysis journal homepage: www.elsevier.com/locate/jpba
Simultaneous analysis of antibiotics in biological samples by ultra high performance liquid chromatography–tandem mass spectrometry Rocío Cazorla-Reyes 1 , Roberto Romero-González 1 , Antonia Garrido Frenich 1,∗ , Manuel Angel Rodríguez Maresca 2 , José Luis Martínez Vidal 1 1 2
Research Group “Analytical Chemistry of Contaminants”, Department of Chemistry and Physics, University of Almería, E-04120 Almería, Spain Torrecardenas Hospital, Almería, E-04009 Almería, Spain
a r t i c l e
i n f o
Article history: Received 17 May 2013 Received in revised form 31 October 2013 Accepted 1 November 2013 Available online 15 November 2013 Keywords: Antibiotics Human biological samples Simple extraction Ultra-high performance liquid chromatography Tandem mass spectrometry
a b s t r a c t A rapid and reliable multiclass method was developed for the simultaneous analysis of 21 antibiotics (beta-lactams, aminoglycosides, penicillins, cephalosporins, carbapenems or quinolones) in urine, serum, cerebrospinal fluid (CSF) and bronchial aspirations by ultra-high performance liquid chromatography coupled to tandem mass spectrometry (UHPLC–MS/MS). Prior to chromatographic determination, the analytes were extracted from human biological fluids by simple sample treatments, which imply dilution, liquefaction, or protein precipitation. Several chromatographic conditions were optimized in order to obtain a fast separation ( 324.4 (15) 586.7 > 163.2 (30) 725.6 > 144.2 (15) 300.3 > 142.0 (25) 481.4 > 396.3 (15) 366.2 > 349.4 (10) 586.6 > 569.8 (20) 301.4 > 168.2 (12) 198.1 > 136.2 (5) 406.4 > 317.4 (20) 547.3 > 468.5 (10) 232.1 > 140.2 (12) 362.3 > 318.6 (20) 555.5 > 396.4 (10) 350.2 > 106.1 (15) 402.5 > 384.6 (20) 338.2 > 296.5 (20) 941.2 > 316.5 (15) 540.4 > 398.5 (20) 749.1 > 158.2 (30) 811.2 > 159.2 (40)
468.7 > 145.2 (20) 586.7 > 264.4 (25) 725.6 > 100.1 (20) 300.3 > 126.2 (20) 481.4 > 324.3 (17) 366.2 > 114.1 (20) 586.6 > 513.6 (22) 301.4 > 92.1 (25) 198.1 > 108.1 (10) 406.4 > 276.3 (20) 547.3 > 167.2 (25) 232.1 > 188.3 (10) 362.3 > 221.3 (35) 555.5 > 167.2 (25) 350.2 > 114.1 (30) 402.5 > 364.6 (25) 338.2 > 235.4 (20) 941.2 > 204.2 (20) 540.4 > 182.1 (25) 749.1 > 590.7 (20) 811.2 > 341.6 (25)
a
Collision energies (eV) are given in brackets.
(a) Urine: 0.5 mL of urine samples was diluted with 1 mL of mobile phase (1:2, v/v). The mixture was vigorously shaken prior to UHPLC–MS/MS analysis. (b) Serum: 1 mL of serum was mixed with 1 mL of acetronitrile. After vigorous shaking, samples were centrifuged at 5000 rpm (4136 × g) for 5 min, and then, 250 L of supernatant was taken and diluted with 750 L of mobile phase (1:3, v/v) before chromatographic analysis. (c) CSF: For the treatment of CSF samples, 800 L of mobile phase was added to 200 L of CSF (1:4, v/v) and they were vigorously shaken. After that, they were injected directly into chromatographic system. (d) Bronchial aspirations: 4 mL of an aqueous solution of DTT (1%, w/v) was added for each gram of sample. After that, samples were vigorously shaken and they were centrifuged at 5000 rpm (4136 × g) for 5 min. Then 1 mL of supernatant was taken and injected directly into UHPLC–MS/MS. 3. Results and discussion 3.1. MS/MS analysis The optimization of MS/MS parameters (cone voltage and collision energy) was performed by infusion of a standard solution of 20 mg/L of each antibiotic in a mixture of methanol:water (50:50, v/v) and 50 L of formic acid (except for sulbactam an clavulanic acid) at a flow rate of 10 L/min. The compounds were injected into the ESI source in positive ion mode at different voltages, except for sulbactam and clavulanic acid, which were monitored in negative ion mode ([M−H]− ). Under the experimental conditions, protonated molecules ([M+H]+ ) were obtained in all the cases, except for meropenem and piperacillin, which formed sodium adduct (m/z 406.4 and m/z 540.4, respectively). For vancomycin, despite molecular weight was 1449.2, the monitored ion was m/z 725.6, because this compound has multiple basic ionizable groups and the diprotonated molecule, [M+2H]2+ , provided the most sensitive signal. The same situation was observed for daptomycin, monitoring the ion at m/z 811.2, despite the molecular weight was 1620.7 [39,40]. Then, collision energies were studied in order to find the most abundant product ions, selecting the most sensitive transition for quantification purposes and a second one for confirmation.
Table 1 shows MS/MS transitions as well as cone voltages and collision energy values optimized for the selected compounds. Although non-specific transitions were avoided, for some compounds such as tigecycline, amoxicillin and moxifloxacin, the most sensitive detected product ions correspond with the loss of ammonia [M+H−NH3 ]+ . 3.2. Chromatographic separation Chromatographic conditions were studied in order to achieve the best separation and retention for the analytes, minimizing analysis time. First, several experiments were performed testing different mobile phases consisting of methanol as organic phase and water with ammonium formate (5 mM) or different concentrations of formic acid (0.01, 0.05 and 0.1%, v/v). The addition of formic acid provided better results than ammonium formate, and it improved the ionization efficiency. Furthermore, 0.01% (v/v) of formic acid provided better peak shape, and therefore methanol, as organic modifier, and an aqueous solution of formic acid 0.01%, v/v were used as mobile phase. Several gradient profiles were studied, and good response was obtained with the gradient described in Section 2.2. Other parameters such as column temperature, flow rate and injection volume were studied in order to get a fast and reliable separation, and the best results were obtained when 30 ◦ C was used as column temperature, 0.3 mL/min as flow rate and 5 L were injected. Under these conditions, retention time of the antibiotics was constant, ranging from 0.61 min (tobramycin) to 3.82 min (daptomycine). The analytes were distributed in seven overlapping acquisition functions, using a maximum of 4 antibiotics (8 transitions) per function. Because there are several compounds detected in positive ion mode that co-eluted with clavulanic acid, which was detected in negative ion mode, polarity switching was not an option and therefore, two injections were carried out, one to monitor the compounds detected in positive ion mode (see Table 1) and the second one to monitor clavulanic acid and sulbactam. Finally, it must be highlighted that one the main advantages of UHPLC is that it is able to perform a run in 6.0 min (positive mode) and 5.8 min (negative mode), minimizing the running time necessary for the detection of the selected compounds, and therefore, it
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Fig. 1. UHPLC–MS/MS chromatograms obtained from a blank serum sample spiked at 1 mg/L.
can be used in routine analysis. Fig. 1 shows a representative chromatogram obtained from a spiked standard mixture at 1 mg/L of the selected antibiotics in serum. 3.3. Optimization of the extraction procedure Extraction procedure is the critical step, when a multiclass antibiotic method has to be developed, due to the different physicochemical properties of the compounds that have to be extracted simultaneously. Generally, antibiotic extraction from biological fluids includes laborious stages of evaporation, elution or cleanup [36]. However, single extraction procedures should be developed for the simultaneous extraction of antibiotics, which cover a wide range of polarity in urine, serum, CSF and bronchial aspirations, in order to reduce sample handling and increase sample throughput. For urine matrix, a simple dilution of sample was investigated. For that, several volumes of spiked urine (2 mg/L) were diluted with mobile phase (1:1 (v/v) of an aqueous solution of formic acid 0.01% (v/v) and methanol). Dilutions 1:1, 1:2, 1:4, 1:9 and 1:100 (v/v) (sample-mobile phase) were tested. It was noted that the more diluted the sample was, less matrix effect was observed; however, the sensitivity was lower. In this sense, better results were obtained when a dilution factor of 1:2 was used, taking into account that this dilution factor provided good sensitivity and minimized the presence of interferences, as well as suitable peak shape was obtained. In the case of serum, 1 mL of acetonitrile was added to 1 mL of spiked sample (2 mg/L) in order to precipitate the proteins present in the matrix. After that, a centrifugation step was carried out. It was observed that 5000 rpm (4136 × g) should be used to get a clean supernatant layer. In order to reduce the burden of matrix
and eliminate the presence of interferences, several dilutions with mobile phase, were evaluated, including 1:1, 1:3 and 1:9 (v/v). Better results were obtained when a dilution factor of 1:3 (v/v) was employed in terms of sensitivity and matrix effect. Finally, a SPE procedure was evaluated in order to improve the sensitivity, bearing in mind that target analytes could be concentrated during the extraction stage. For that purpose, two types of cartridges, Oasis HLB and C18, were evaluated, and they were conditioned with 5 mL of methanol and 5 mL of water. After that, the diluted supernatant was passed through the cartridges. They were dried during 30 min and target compounds were eluted using a mixture of methanol:water (1:1, v/v). When this procedure was applied, recoveries did not improve, and therefore, it was not applied in further experiments. For CSF, spiked samples (2 mg/L) were diluted with mobile phase (1:1, 1:4, 1:9 (v/v)). The best recoveries were obtained when samples were diluted 1:4 (v/v). Moreover, suitable response was achieved when this dilution was used. Different volumes (0, 50 and 100 L) of a solution of phosphate buffer 0.1 M at pH 7, which is a system for regulation of intra and extra cellular pH [41], were added to CSF samples in order to homogenize the tissue, but recoveries were not improved when these experiments were performed, and therefore, it was not used for further experiments. Finally, the extraction method of antibiotics from bronchial aspirations was optimized. First, 4 mL of an aqueous solution of DTT (1%, w/v) was added to 1 g of spiked sample (2 mg/kg) to liquefy the sample [42]. Then, a centrifugation step at 5000 rpm (4136 × g) for 5 min was carried out and a clean extract was obtained. After that, 500 L of the obtained supernatant was diluted with 500 L of mobile phase (1:1, v/v) and injected into the UHPLC system, and
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Fig. 2. Slope ratios between matrix-matched and solvent calibration of the target antibiotics. Compliance interval covering the range between 0.8 and 1.2 for tolerable matrix effect has been plotted in: (a) urine; (b) serum; (c) CSF and (d) bronchial aspiration. Compounds code: (1) tobramycin; (2) amikacin; (3) vancomycin; (4) imipenem; (5) cefepime; (6) amoxicillin; (7) tigecycline; (8) tazobactam; (9) clavulanic acid; (10) meropenem; (11) ceftazidime; (12) sulbactam; (13) levofloxacin; (14) ceftriaxone; (15) ampicillin; (16) moxifloxacin; (17) linezolid; (18) teicoplanin; (19) piperacillin; (20) clarithromycin; (21) daptomycin.
the results were compared with those obtained injecting the supernatant directly. Bearing in mind that the dilution did not improve the results and worst sensitivity was obtained, the supernatant was injected directly into the UHPLC–MS/MS system. 3.4. Evaluation of matrix effect When ESI is used as ionization technique, one of the main problems is the signal suppression or enhancement of the analytes due to the other components present in the matrix. Moreover, biological fluids are very complex matrices, which have a large amount of compounds that can interfere with the target compounds. Therefore, matrix effect was evaluated in order to ensure bias-free analytical results. For that purpose, five concentration levels (from 0.5 to 5 mg/L) were analyzed in solvent (mixture of methanol:water 1:1, v/v) and in extracted blank samples (matrix). Fig. 2 shows slope ratios matrix/solvent for each compound, and a tolerable signal suppression or enhancement effect was considered if the slope ratio ranged from 0.8 to 1.2, whereas lower values than 0.8 or higher than 1.2 implies a strong effect, signal suppression or enhancement respectively. In the case of urine, it was observed that despite the dilution of the sample, there was a strong matrix effect for most of the compounds except for meropenem, sulbactam, linezolid, teicoplanin, clarithromycin and daptomycine. For these compounds, slope ratio was between 0.8 and 1.2. It can be noted that some compounds (tobramycin, amikacin, vancomycin, imipenem, cefepime, amoxicillin, tigecycline, tazobactam, clavulanic acid, ceftazidime, levofloxacin, ampicillin and moxifloxacin) show a matrix suppression effect (slope ratio < 0.8), whereas a signal enhancement was observed for piperacillin and ceftriaxone (slope ratio > 1.2). When serum was evaluated, matrix effect is not observed for levofloxacin, linezolid, teicoplanin and piperacillin. Furthermore, there was a signal enhancement for meropenem, clarithromycin and daptomycine. The rest of the compounds show a matrix suppression effect. For CSF, amoxicillin, meropenem, sulbactam, levofloxacin, ampicillin, moxifloxacin, linezolid and teicoplanin do not present a matrix effect, observing a matrix enhancement effect for cefepime, piperacillin, clarithromycin and daptomycine. For the rest of the compounds, a matrix suppression effect was observed.
Finally, for bronchial aspiration samples, matrix effect is present in all compounds except for ceftazidime, ceftriaxone and ampicillin. It can be observed a matrix enhancement effect for vancomycin, imipenem, cefepime, amoxicillin, tigecycline, tazobactam, piperacillin and clarithromycin, whereas for the other compounds, a matrix suppression effect is exhibited. Because the existence of matrix effect, despite the dilutions performed in the four matrices evaluated, matrix matched calibration was used for quantification purposes during the determination of the target compounds in samples. 3.5. Validation Performance characteristics of the optimized methods were established by a validation procedure with blank urine, serum, CSF and bronchial aspiration samples, studying selectivity, linearity, limits of detection (LODs) and quantification (LOQs), trueness, repeatability and interday precision. First, a stability study of the compounds in the assayed matrices was carried out spiking blank matrices at 1 mg/L (1 mg/kg in case of bronchial aspiration matrix). The samples were stored at −80 ◦ C and −30 ◦ C for 20 days, by measuring the bias compared to the theoretical concentration (maximum bias accepted ±20%), and the samples were analyzed weekly. It was observed that the compounds were stable for one week if they are stored at −80 ◦ C, whereas at −30 ◦ C, the bias was higher than 20% for some compounds, such as tigecycline, meropenem, clavulanic acid, amoxicillin and ceftriaxone. Bearing in mind that in routine analysis, the results should be provided within 24 h, the samples were stored at −30 ◦ C before analysis. Besides, a short-term stability was also performed, observing that it was guaranteed up to 8 h, if the samples were stored at 8 ◦ C in the autosampler. Method linearity was assayed by means of matrix-matched standard calibration, preparing one calibration curve for every matrix, spiking blank samples with the selected antibiotics in the range from 0.1 to 5 mg/L (from 0.1 to 5 mg/kg for bronchial aspiration matrix). Calibration graphs were obtained by least-squares linear regression analysis, using peak area as analytical response. The responses were linear in the assayed range and determination coefficient (R2 ) values were higher than 0.970, 0.988, 0.970 and 0.990, for urine, serum, CSF and bronchial aspirations, respectively.
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Table 2 Validation parameters of the optimized UHPLC–MS/MS method in urine. Antibiotic
LOD (mg/L)
LOQ (mg/L)
Tobramycin Amikacin Vancomycin Imipenem Cefepime Amoxicillin Tigecycline Tazobactam Clavulanic acid Meropenem Ceftazidime Sulbactam Levofloxacin Ceftriaxone Ampicillin Moxifloxacin Linezolid Teicoplanin Piperacillin Clarithromycin Daptomycin
0.30 0.30 0.30 0.25 0.10 0.10 0.30 0.30 0.10 0.10 0.25 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.25
1.00 1.00 1.00 0.50 0.50 0.50 1.00 1.00 0.50 0.50 0.50 0.50 0.50 0.50 0.50 0.50 0.50 0.50 0.50 0.50 0.50
Recoverya 0.5 mg/L
a b c
–c – – 75 (7) 75 (24) 110 (15) – – 76 (16) 75 (19) 76 (18) 91 (17) 79 (9) 115 (7) 70 (10) 88 (14) 110 (13) 77 (21) 107 (7) 116 (6) 79 (13)
Interday precisionb 1 mg/L
2 mg/L
70 (20) 71 (19) 74 (17) 75 (8) 84 (7) 72 (10) 72 (10) 74 (12) 90 (6) 97 (16) 92 (10) 82 (5) 79 (7) 89 (3) 71 (8) 88 (3) 79 (11) 78 (14) 98 (3) 74 (5) 86 (10)
71 (14) 72 (16) 71 (15) 80 (5) 77 (7) 71 (6) 70 (5) 70 (10) 89 (5) 85 (13) 84 (4) 88 (2) 71 (7) 73 (3) 75 (10) 71 (2) 70 (7) 72 (11) 77 (2) 71 (3) 78 (9)
10 22 21 8 13 18 16 13 7 24 14 10 11 13 16 11 15 18 20 6 7
RSD values of repeatability are given in brackets (n = 5). RSD values obtained at 1 mg/L (n = 5). Not quantified at this concentration.
Deviation of the residuals of each calibration point was also studied, checking it was always equal or lower than 20%. For those samples with concentrations higher than the upper linear concentration range, a dilution with mobile phase was carried out to get a final concentration within the linear range of the method. The sample and the blank sample used for calibration purposes were submitted to this elution step. LODs and LOQs were calculated by analyzing blank samples spiked at 0.01, 0.05, 0.1, 0.2, 0.5, and 1 mg/L (at 0.01, 0.05, 0.1, 0.2, 0.5 and 1 mg/kg for bronchial aspiration samples) and they were determined as the lowest concentration of analyte for which signalto-noise ratios were 3 and 10, respectively (Tables 2–5). LODs were always equal or lower than 0.3 mg/L, whereas LOQs ranged from
0.50 to 1.00 mg/L, from 0.04 to 1.00 mg/L, from 0.01 to 1.00 mg/L and from 0.01 to 0.66 mg/kg in urine, serum, CSF and bronchial aspirations, respectively. It can be observed that lower limits were obtained in CSF, which can be considered a “clean matrix”, and in bronchial aspirations, indicating that the use of DTT is effective for the determination of antibiotics in this type of matrices. Furthermore it can be highlighted that bearing in mind therapeutic doses, the sensitivity of the proposed method was enough to detect the presence of the selected compounds in the assayed matrices. In order to evaluate the trueness of the proposed method, recovery studies were carried out by fortifying blank samples of each matrix with antibiotics at three concentration levels (0.5, 1 and 2 mg/L (0.5, 1 and 2 mg/kg in case of bronchial aspirations)),
Table 3 Validation parameters of the optimized UHPLC–MS/MS method in serum. Antibiotic
LOD (mg/L)
LOQ (mg/L)
Tobramycin Amikacin Vancomycin Imipenem Cefepime Amoxicillin Tigecycline Tazobactam Clavulanic acid Meropenem Ceftazidime Sulbactam Levofloxacin Ceftriaxone Ampicillin Moxifloxacin Linezolid Teicoplanin Piperacillin Clarithromycin Daptomycin
0.04 0.04 0.10 0.40 0.40 0.40 0.30 0.30 0.10 0.20 0.04 0.10 0.04 0.50 0.04 0.04 0.10 0.02 0.04 0.01 0.20
0.10 0.15 0.40 0.20 0.20 0.20 1.00 1.00 0.20 0.50 0.10 0.20 0.10 1.00 0.10 0.10 0.25 0.10 0.15 0.04 0.50
Recoverya 0.5 mg/L
a b c
RSD values of repeatability are given in brackets (n = 5). RSD values obtained at 1 mg/L (n = 5). Not quantified at this concentration.
111 (8) 108 (9) 104 (16) 95 (2) 102 (17) 77 (23) –c – 72 (23) 109 (18) 105 (24) 89 (5) 84 (4) – 77 (13) 95 (3) 76 (8) 77 (22) 74 (18) 70 (11) 91 (24)
Interday precisionb 1 mg/L 74 (11) 86 (5) 100 (10) 83 (5) 71 (15) 81 (12) 70 (20) 71 (20) 73 (4) 84 (10) 90 (14) 95 (4) 76 (2) 75 (20) 87 (6) 80 (2) 80 (4) 78 (13) 73 (14) 76 (5) 101 (20)
2 mg/L 78 (6) 77 (1) 72 (8) 86 (4) 74 (8) 91 (8) 71 (19) 70 (15) 78 (1) 75 (2) 106 (13) 89 (1) 75 (2) 78 (15) 81 (2) 74 (4) 90 (5) 117 (12) 76 (7) 75 (3) 72 (12)
11 5 16 5 20 14 22 20 7 6 14 4 2 23 6 2 4 20 14 8 13
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Table 4 Validation parameters of the optimized UHPLC–MS/MS method in cerebrospinal fluid (CSF). Antibiotic
LOD (mg/L)
LOQ (mg/L)
Tobramycin Amikacin Vancomycin Imipenem Cefepime Amoxicillin Tigecycline Tazobactam Clavulanic acid Meropenem Ceftazidime Sulbactam Levofloxacin Ceftriaxone Ampicillin Moxifloxacin Linezolid Teicoplanin Piperacillin Clarithromycin Daptomycin
0.020 0.300 0.100 0.010 0.142 0.007 0.087 0.300 0.038 0.200 0.001 0.003 0.016 0.064 0.148 0.001 0.002 0.300 0.001 0.001 0.028
0.067 1.000 0.300 0.035 0.474 0.024 0.291 1.000 0.128 0.500 0.003 0.010 0.053 0.215 0.049 0.003 0.007 1.000 0.019 0.001 0.094
Recoverya 0.5 mg/L
a b c
104 (21) –c 70 (20) 90 (19) 110 (13) 93 (3) 89 (21) – 76 (17) 70 (24) 106 (6) 101 (7) 75 (9) 72 (21) 103 (19) 112 (10) 102 (2) – 90 (10) 108 (4) 107 (3)
Interday precisionb 1 mg/L 104 (17) 73 (24) 71 (13) 102 (13) 95 (9) 97 (7) 75 (14) 71 (24) 79 (14) 78 (22) 116 (8) 111 (6) 88 (4) 73 (15) 106 (7) 113 (2) 112 (3) 70 (20) 84 (12) 113 (2) 104 (4)
2 mg/L 1012 (15) 70 (14) 72 (10) 108 (13) 109 (8) 98 (8) 70 (11) 76 (19) 99 (3) 74 (15) 115 (10) 101 (3) 107 (4) 71 (13) 106 (9) 103 (2) 111 (3) 97 (18) 77 (11) 109 (2) 98 (4)
14 19 13 12 24 11 11 19 24 20 14 7 5 14 9 5 6 21 13 5 5
RSD values of repeatability are given in brackets (n = 5). RSD values obtained at 1 mg/L (n = 5). Not quantified at this concentration.
performing 5 replicates at each level (Tables 2–5), observing that adequate recoveries (values ranging from 70 to 120%) were obtained for all the compounds in the four matrices evaluated. Thus, for urine, the obtained values ranged from 70% (ampicillin) to 116% (clarithromycin) for 0.5 mg/L, 70% (tobramycin) to 98% (piperacillin) for 1 mg/L and 70% (tigecycline and tazobactam) to 89% (clavulanic acid) for 2 mg/L. Tobramycin, amikacin, vancomycin, tygecillin and tazobactam were not quantified at 0.5 mg/L in urine matrix. For serum matrix, at 0.5 mg/L, the recoveries ranged from 70% (clarithromycin) to 111% (tobramycin), at 1 mg/L recovery values ranged from 70% (tigecycline) to 101% (daptomycin) and from 70%
(tazobactam) to 117% (teicoplanin) when the samples were spiked with 2 mg/L. In this case, tigecycline, tazobactam and ceftriaxone could not be quantified at 0.5 mg/L. In the case of CSF, the obtained results showed that for 0.5 mg/L, the recoveries ranged from 70% (meropenem) to 112% (moxifloxacin), for 1 mg/L recovery values ranged from 70% (teicoplanin) to 116% (ceftazidime) and from 70% (tigecycline and amikacin) to 115% (ceftazidime) when the samples were spiked with 2 mg/L. The analytes amikacin, tazobactam and teicoplanin could not be quantified at 0.5 mg/L. For bronchial aspirations, the obtained recoveries ranged from 70 (teicoplanin and tazobactam) to 107% (amoxicillin and
Table 5 Validation parameters of the optimized UHPLC–MS/MS method in bronchial aspiration. Antibiotic
LOD (mg/kg)
LOQ (mg/kg)
Tobramycin Amikacin Vancomycin Imipenem Cefepime Amoxicillin Tigecycline Tazobactam Clavulanic acid Meropenem Ceftazidime Sulbactam Levofloxacin Ceftriaxone Ampicillin Moxifloxacin Linezolid Teicoplanin Piperacillin Clarithromycin Daptomycin
0.013 0.027 0.200 0.001 0.002 0.001 0.200 0.002 0.005 0.200 0.200 0.006 0.008 0.196 0.020 0.001 0.200 0.040 0.013 0.005 0.036
0.045 0.090 0.500 0.004 0.006 0.003 0.167 0.008 0.018 0.500 0.667 0.006 0.027 0.655 0.068 0.002 0.667 0.133 0.043 0.016 0.120
Recoverya 0.5 mg/kg
a b c
RSD values of repeatability are given in brackets (n = 5). RSD values obtained at 1 mg/kg (n = 5). Not quantified at this concentration.
94 (13) 102 (20) 87 (18) 106 (15) 100 (18) 107 (11) 111 (17) 70 (20) 71 (23) 97 (23) 106 (18) 105 (3) 107 (16) –c 105 (20) 104 (13) – 70 (20) 105 (17) 106 (12) 105 (7)
Interday precisionb 1 mg/kg
2 mg/kg
90 (3) 109 (16) 82 (14) 102 (3) 91 (9) 119 (4) 79 (12) 74 (15) 72 (13) 84 (17) 94 (10) 81 (2) 101 (6) 107 (12) 102 (7) 100 (1) 78 (3) 77 (14) 110 (5) 112 (3) 104 (4)
99 (1) 72 (10) 87 (13) 76 (3) 73 (9) 78 (4) 72 (9) 79 (11) 71 (14) 70 (12) 101 (8) 79 (3) 73 (9) 77 (10) 76 (13) 75 (1) 90 (6) 95 (12) 74 (5) 77 (2) 80 (8)
5 21 17 5 17 8 12 13 18 18 13 4 10 19 9 4 5 16 10 9 7
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Table 6 Concentration of antibiotics found in real samples from ten patients.a Patient
Antibiotic
Urine
Serum
CSF
Bronchial aspirations
P1
Amoxicillin Clavulanic acid
122.8 22.5
0.7 1.0
