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MS method for the determination of teicoplanin in human plasma
Journal of Chromatography B, 1008 (2016) 125–131
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Journal of Chromatography B journal homepage: www.elsevier.com/locate/chromb
Direct injection LC–MS/MS method for the determination of teicoplanin in human plasma Kwang-Youl Kim a,1 , Sang-Heon Cho a,1 , Yong-Hyun Song a , Moon-Suk Nam a,b , Cheol-Woo Kim a,b,∗ a b
Department of Clinical Pharmacology, Inha University Hospital, Inha University School of Medicine, Incheon, South Korea Department of Internal Medicine, Inha University Hospital, Inha University School of Medicine, Incheon, South Korea
a r t i c l e
i n f o
Article history: Received 3 September 2015 Received in revised form 19 November 2015 Accepted 20 November 2015 Available online 24 November 2015 Keywords: Teicoplanin Multiple reaction monitoring Direct injection Therapeutic drug monitoring
a b s t r a c t A direct injection-based, simple, accurate, and robust LC–MS/MS method was developed and validated for the determination of teicoplanin in human plasma. Patient plasma samples were diluted in an aqueous buffer prior to injection into the LC–MS/MS system. Chromatographic separation was achieved using a Cadenza HS-C18 column and a gradient mixture of acetonitrile–water (both containing 0.1% formic acid) as the mobile phase at a flow rate of 0.5 mL/min. The analytes were detected in multiple reaction monitoring mode with positive ion electrospray ionization. The concentration of teicoplanin was determined as the sum of six components (A3-1, A2-1, A2-2, A2-3, A2-4, and A2-5). The calibration curve was linear over a concentration range of 1–50 mg/L, which covered the clinically accepted trough and therapeutic plasma levels. The intra- and inter-day precision and accuracy values were both less than 15%. This validated method was successfully applied to therapeutic drug monitoring of teicoplanin in routine clinical practice. Thus, we expect it to be useful for the determination of teicoplanin concentration in human plasma. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Teicoplanin is a glycopeptide antibiotics produced by Actinoplanes teichomyceticus and is commonly used to treat infections caused by gram-positive bacteria including methicillin-resistant Staphylococcus aureus (MRSA) and Enterococcus faecalis [1,2]. It comprises five major components (A2-1, A2-2, A2-3, A2-4, and A25), one hydrolysis component (A3-1), and four minor components (RS-1–RS-4). The main component is an A2 mixture that forms about 90–95% of teicoplanin. The A2-2 component is the most active compound and constitutes over 50% of the A2 mixture [3]. The concentration of teicoplanin in plasma and serum is generally determined through fluorescence polarization immunoassay (FPIA) and HPLC analysis. However, both FPIA and HPLC methods are often time consuming and cumbersome for sample pretreatment or analysis procedures [4–8]. In clinical laboratories, liquid chromatography–tandem mass spectrometry (LC–MS/MS)
∗ Corresponding author at: Department of Clinical Pharmacology, Inha University Hospital, Inha University School of Medicine, Incheon, South Korea. Fax: +82 32 882 6578. E-mail address: [email protected] (C.-W. Kim). 1 These authors contributed equally on this paper. http://dx.doi.org/10.1016/j.jchromb.2015.11.037 1570-0232/© 2015 Elsevier B.V. All rights reserved.
has become a popular assay to measure the levels of drugs, metabolites, peptides, and proteins of interest in complex biological samples owing to its excellent sensitivity and selectivity [9–11]. Recently, several LC–MS/MS methods have been reported for the quantification of teicoplanin in plasma samples. Fung et al. developed an LC–MS/MS method in which only two components (A2-2 & A2-3 isoforms) of teicoplanin were used to measure its concentration in plasma [12]. Tsai et al. described an UPLC–MS/MS method to simultaneously quantify the concentrations of six teicoplanin components (A3-1, A2-1–A2-5), including three antibiotics (vancomycin, daptomycin, and colistin), in human plasma [13]. Mueller et al. presented a high-resolution LC–MS method with online extraction based on turbulent flow chromatography for quantification of teicoplanin by measuring the five major components (A2-1–A2-5) [14]. With respect to sample preparation, the abovementioned methods mainly used protein precipitation with organic solvents. However, organic solvent-based protein precipitation often requires multistep procedures such as further dilution and the consumption of large amounts of solvent and the use of mixtures of several solvents for recovery and reduction of matrix effects [12–14]. Therefore, a more simple method for the preparation of plasma samples is required.
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It has recently been reported that a simple, direct-injection HPLC-based method using a novel hydrophobic/hydrophilic hybrid C18 column can effectively remove proteins in aqueous solutions [15]. Thus, our objective was to reduce the sample preparation procedure and develop an efficient LC–MS/MS method for teicoplanin quantification using a hybrid C18 column. In this manuscript, we report a validated LC–MS/MS method using direct injection for quantification of teicoplanin by measuring its six components (A3-1, A2-1–A2-5) in human plasma. This method is simple and robust, making it suitable for therapeutic drug monitoring (TDM) of teicoplanin in routine clinical practice.
Mass spectrometric detection was performed with an API 4000 mass spectrometer (MDS SCIEX, Toronto, Canada) operated in positive ion electrospray ionization (ESI) mode. The source parameters of the mass spectrometer were optimized and maintained as follows: collision activated dissociation (CAD) gas, 5; curtain gas (CUR), 20; gas 1 (nebulizer gas), 60; gas 2 (heater gas), 60; turbo ion spray (IS) voltage, 5300 V; source temperature, 450 ◦ C. Nitrogen was used as the nebulizer, auxiliary, curtain, and collision gases. Data processing was performed on Analyst 1.5.1 software (SCIEX).
2. Experimental
The analytical method for teicoplanin in human plasma was validated on the basis of the following parameters: specificity, linearity, lower limit of quantification (LLOQ), precision, accuracy, matrix effect, and sample stability.
2.1. Materials and reagents Teicoplanin standard (CAS Number 91032-26-7), internal standard (IS, sulfamethoxazole, CAS Number 723-46-6) (Fig. 1), and ACS reagent-grade formic acid were purchased from Sigma–Aldrich (St. Louis, MO, USA). HPLC-grade acetonitrile and methanol were obtained from Burdick and Jackson (Muskegon, MI, USA). Water was filtered using a Millipore Milli-Q system (Milford, MA, USA). Blank (drug-free) human plasma was obtained from the Clinical Trial Center at Inha University Hospital (Incheon, South Korea). 2.2. Preparation of standard and quality control (QC) samples Stock solutions of teicoplanin and IS were prepared in 50% methanol at concentrations of 10 mg/mL and stored at −20 ◦ C until dilution. Teicoplanin working solutions at concentrations of 10, 20, 50, 100, 250, and 500 g/mL were prepared via serial dilution with water. Sulfamethoxazole was diluted with 0.1% formic acid in water to prepare a stock solution at a final concentration of 500 ng/mL. Quality control (QC) working solutions were prepared at 30, 200, and 400 g/mL. All standard and quality control working solutions were kept at 4 ◦ C before analysis. Drug-free blank plasma was spiked with teicoplanin working solutions to prepare a calibration curve of 1, 2, 5, 10, 25, and 50 g/mL. In addition, QC samples for teicoplanin were prepared in drug-free plasma at concentrations of 3 g/mL (QCL; low), 20 g/mL (QCM; medium), and 40 g/mL (QCH; high) in the same manner as the calibration curves. All samples were prepared daily. 2.3. Sample preparation All samples (20 L) were added to the IS (20 L, 500 ng/mL in 0.1% formic acid solution) and diluted with 200 L of water containing 0.1% formic acid. The sample mixtures were vortexed and centrifuged for 5 min at 13,000 rpm, and the supernatant (1 L) was injected on the LC–MS/MS for analysis. 2.4. Liquid chromatography and mass spectrometry Chromatographic separation was performed on an Agilent 1200 series HPLC System (Santa Rosa, CA, USA) consisting of a binary Pump (G1312A) and an autosampler (G1367B) with thermostat (G1330B) equipped with a Cadenza HS-C18 analytical column (75 mm × 3.0 mm i.d., 3 m) maintained at 40 ◦ C. The mobile phase comprised water with 0.1% formic acid (mobile phase A) and 0.1% formic acid in acetonitrile (mobile phase B). The flow rate was 0.5 mL/min with the following gradient conditions: 0–1 min, 0% B (isocratic); 1–2 min, 0–100% B (linear gradient); 2–3 min, 100% B (isocratic); 3–3.5 min, 100–0% B (linear gradient); 3.5–6.5 min, 0% B (isocratic). Total run time was 6.5 min with 1 L injection. Autosampler temperature was set at 10 ◦ C.
2.5. Method validation
2.5.1. Specificity Specificity was evaluated by analyzing six different drug-free plasma samples to investigate potential interference from coeluting endogenous compounds in the LC peak region of teicoplanin and IS. 2.5.2. Linearity and lower limit of quantification Calibration curves were constructed by plotting the peak area ratios of a combination of six teicoplanin components to the IS vs. the nominal concentration. Linearity was tested with teicoplanin concentrations of 1–50 g/mL (1, 2, 5, 10, 25, 50 g/mL). The regression parameters of slope and y-intercept and correlation coefficient values were calculated by linear least-squares regression analysis using 1/X as the weighting factor. The LLOQ was the lowest concentration on the calibration curve that could be quantified with an acceptable precision of less than 20% and accuracy within ±20%, which was evaluated using five replicate samples. 2.5.3. Precision and accuracy The intra-day accuracy and precision of the assay were determined by analyzing three QC samples (n = 5) on the same day. The inter-day accuracy and precision were evaluated by the analysis of each QC sample over five consecutive days. Precision was determined on the basis of the coefficient of variation (C.V. (%)), and the accuracy was calculated as relative error (R.E. (%)). The acceptance criteria for precision and accuracy should be within ±15% compared to the nominal values. 2.5.4. Matrix effect and recovery The matrix effect was evaluated (n = 5) by comparing the peak areas of teicoplanin solutions spiked with diluted blank plasma samples and the same neat solutions as the three QC samples. The recovery was measured (n = 5) by comparing the peak areas of three QC samples and teicoplanin neat solution at these levels. The matrix effect and recovery of the IS was also measured using the same method. 2.5.5. Stability The stability test was performed (n = 3) with three QC samples under the following conditions: short-term and long-term temperature stability and freeze and thaw stability. 2.6. Analysis of patient samples For the determination of teicoplanin concentration in human plasma, all patient samples were centrifuged at 3000 rpm at 10 ◦ C for 10 min and stored at −70 ◦ C until analysis. The samples
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Fig. 1. Chemical structures of the major components (A2-1–A2-5) of teicoplanin and the internal standard (sulfamethoxazole).
Table 1 Optimized MS/MS conditions of six teicoplanin components and the internal standard.
Teicoplanin A2-1 Teicoplanin A2-2 & A2-3 Teicoplanin A2-4 & A2-5 Teicoplanin A3-1 Sulfamethoxazole (IS)
MRM transitions
DP (V)
CE (V)
CXP (V)
EP (V)
939.7/314.2 940.7/316.2 947.8/330.2 782.4/203.7 254.2/92.0
60 60 60 60 60
15 16 17 15 25
10 10 16 8 12
10 10 10 10 10
MRM, multiple reaction monitoring; DP, declustering potential; CE, collision energy; CXP, collision cell potential energy; EP, entrance potential.
were prepared using the above dilution method and subjected to LC–MS/MS analysis. 3. Results and discussion 3.1. Mass spectrometry We investigated the m/z values of the precursor ions for six components of teicoplanin and the IS in positive ion ESI mode through direct infusion and estimated their product ions. As a result, the doubly charged molecular ions [M + 2H]2+ of the teicoplanin components were predominantly detected in the acidic aqueous mobile phase due to the structure of the polypeptide moiety. The precursor ions of teicoplanin A3-1, A2-1, A2-2 and A2-3 (isomer), and A2-4 and A2-5 (isomer) were the doubly protonated molecular ions [M + 2H]2+ at m/z 782.4, 939.7, 940.7, and 947.8, respectively (Fig. 2). The MS/MS spectra for the six components were obtained with ramped collision energy. The product ions of teicoplanin A31, A2-1, A2-2 and A2-3 (isomer), and A2-4 and A2-5 (isomer) were abundantly produced at m/z 204.1, 314.2, 316.2, and 330.2, respectively (Fig. 2). The IS produced a protonated precursor ion [M + H]+ at m/z 254.2 and a major product ion at m/z 92.0. These major product ions were selected for multiple reaction monitoring (MRM) analysis, for which the dwell time was set to 50 ms. Detailed compound parameters are summarized in Table 1.
3.2. Liquid chromatography To obtain good chromatographic separation for teicoplanin components and the IS, we investigated mobile phase compositions with water, acetonitrile, methanol, and various pH conditions. Acetonitrile was chosen as the organic solvent as it resulted in appropriate sensitivity and peak shape of the teicoplanin components. Good ionization and fragmentation of the teicoplanin components was achieved in the presence of 0.1% formic acid. Therefore, 0.1% formic acid was included in both water and acetonitrile as mobile phase A and B, respectively. The separation of teicoplanin components and the IS was conducted with a gradient solvent system using a Cadenza HS-C18 column with dimensions of 75 mm × 3.0 mm i.d. and 3 m particle size. The initial composition was 0% solvent B for 1 min, increased to 100% solvent B over 2 min, maintained at 100% solvent B until 3 min, decreased to 0% solvent B at 3.5 min, and maintained at 0% solvent B until the end of the run time at 6.5 min. A representative chromatogram showing typical peak shapes for each analyte is depicted in Fig. 3B. Retention times for teicoplanin A3-1, A2-1, A2-2 and A2-3 (isomer), A2-4 and A2-5 (isomer), and the IS were 3.47, 3.47, 3.48, 3.49, and 3.54 min, respectively. All teicoplanin components including the IS were detected with little overlap after 3.0 min. There was no crosstalk with a dwell time of 50 ms between the MRM channel arising from the IS. Therefore, analysis is relatively quick (within 6.5 min) and reproducible without the need for further chromatographic separation of teicoplanin components. It is desirable to use a stable isotope-labeled analog of the analyte molecule as the internal standard in a quantitative MRM-MS assay. However, such compounds for teicoplanin are not commercially available. To select the proper IS on the basis of a similar retention time and matrix effect for teicoplanin components, other drugs were injected into our LC–MS/MS system. As a result, the sulfamethoxazole was selected as the most proper IS in this study because of its similar retention time (3.54 min, Fig. 3B) and matrix effect (100.52 ± 2.06%, Table 4) and the stability of ionization.
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Fig. 2. Collision-induced dissociation mass spectrum of each teicoplanin component and the IS in positive ion electrospray ionization mode. Product-ion spectrum from (a) A2-1, (b) A2-2 and A2-3, (c) A2-4 and A2-5, and (d) A3-1 obtained by fragmentation of [M + 2H]2+ at each m/z and (e) IS [M + H]+ at m/z 254.2 using a collision potential of 20 eV. The MRM transitions of the teicoplanin component and IS that produced the highest product-ion (P+ ) signals were used for teicoplanin quantification.
Table 2 Linearity data for the analysis of teicoplanin in plasma (n = 5). Nominal concentration (g/mL)
Calculated concentration (g/mL)
R.E. (%)
C.V. (%)
R2
1 2 5 10 25 50
0.96 1.97 5.05 10.36 25.60 49.08
3.70 1.30 0.92 −3.56 −2.40 1.84
2.63 2.01 3.45 3.94 1.89 1.01
0.99
R.E., relative error. C.V., coefficient of variation. R2 , coefficient of determination.
3.3. Sample preparation Direct injection is a cost-effective and time-saving method of sample preparation and can also decrease the error associated with sample handling, such as pipetting and labeling. However, the biggest drawback for direct injection of plasma samples is that con-
ventional HPLC columns are easily clogged due to the abundance of proteins such as albumin. Recently, analytical columns have been designed to exclude proteins while allowing smaller molecules to interact with unique stationary phases [15]. The Cadenza HS-C18 column has a hybrid ODS stationary phase with hydrophobic and hydrophilic groups, which helps to separate proteins and drugs of interest from biological samples in an aqueous mobile phase. We first investigated the degree to which various polar and nonpolar analytes, including teicoplanin, vancomycin, and other small molecule drugs, are separated from proteins under the above conditions. This revealed that some hydrophobic analytes such as teicoplanin components were well separated from proteins (Fig. 4A). However, some hydrophilic analytes such as vancomycin were too polar to be sufficiently separated from the larger proteins such as albumin (>30 kDa). Therefore, these polar compounds are not suitable for direct injection as they were eluted within two minutes (Fig. 4A). This column provides excellent separation for low molecular weight analytes, especially, hydrophobic compounds, from the dilute plasma proteins. Therefore, we have selected the Cadenza HS-C18 column as the direct
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Fig. 3. Representative MRM chromatograms for six components of teicoplanin and the IS in (A) blank human plasma and (B) 1 g/mL (LLOQ) teicoplanin spiked human plasma.
Fig. 4. (A) Representative chromatograms of nine drugs as standards in buffer solvent. (B) Chromatogram of the separation of diluted plasma proteins using the Cadenza HS-C18 column. The UV/VIS detector was operated in dual wavelength mode at 210 nm and 254 nm. Larger proteins were excluded from the column under these conditions (column, Cadenza HS-C18 (75 mm × 3.0 mm i.d., 3 m); flow rate, 0.5 mL/min; injection volume, 1 L; mobile phase, water/acetonitrile, both containing 0.1% formic acid, under gradient conditions).
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Table 3 Precision and accuracy for the determination of teicoplanin in human plasma (n = 5). Nominal concentration (g/mL)
3 20 40
Intra-day
Inter-day
R.E. (%)
C.V. (%)
R.E. (%)
C.V. (%)
−9.07 −2.24 −0.53
5.71 2.82 1.30
−12.60 −4.80 0.30
7.55 8.42 3.21
R.E., relative error. C.V., coefficient of variation.
injection column for teicoplanin quantification in human plasma samples. We also evaluated the effect of a dilution buffer with various acidic additives, such as formic acid and ammonium formate, while maintaining the minimum sensitivity of teicoplanin through plasma levels of <10 mg/L and minimizing the matrix effects. When plasma samples were diluted 10-fold with deionized water containing 0.1% formic acid, the sensitivity and reproducibility were suitable for the TDM analysis of teicoplanin. Finally, a post-column switching valve was employed to divert the flow from 0 to 3 min to the waste in order to minimize contamination from the eluted plasma proteins in the mass analyzer. The flow was then switched back to the MS detector from 3 to 5 min (Fig. 4B).
3.4.4. Matrix effect and recovery Matrix effect and recovery were investigated at three QC levels, as described above, and the results are summarized in Table 4. These results indicated that ion suppression or enhancement from the plasma matrix was not observed.
3.4.5. Stability The stability results showed that teicoplanin spiked in human plasma was stable for 6 h at room temperature, for 30 days at −70 ◦ C, and during three freeze–thaw cycles. The results of the stability experiments are listed in Table 5. These results demonstrated that teicoplanin is stable under the described conditions.
3.5. Robustness of direct injection methodology The robustness of our direct injection approach was investigated by sequential injections of 1000 diluted plasma samples. Reproducibility (MS sensitivity, peak shape, and retention time) was shown to be constant and column pressure was less than 6% RSD (Fig. 5). Therefore, this direct injection method has potential clinical applications for high-throughput analysis, especially, for hydrophobic analytes.
3.4. Method validation 3.6. Application 3.4.1. Specificity No interference peaks from the endogenous matrix sample were observed with peaks of the teicoplanin components and the IS (Fig. 3A). Blank plasma samples spiked with teicoplanin at the LLOQ and IS showed typical chromatograms (Fig. 3B). 3.4.2. Linearity and lower limit of quantification The calibration curves ranging from 1–50 g/mL for the sum of all six teicoplanin components in human plasma were performed on five consecutive days and the back-calculated values for each level were measured. As shown in Table 2, the calibration curves of the sum of all six teicoplanin components showed good linearity over the analysis range, with the observed deviation being within ±15% and coefficient of determination (r2 ) above 0.99 for all calibration concentrations. The response at the LLOQ was 10 times greater than the response of blank plasma. Moreover, the values of R.E. and C.V. (n = 5) for LLOQ were 3.7% and 2.63%, respectively, demonstrating that this method is sensitive enough to determine the level of teicoplanin in plasma. 3.4.3. Precision and accuracy As shown in Table 3, the intra-day precision and accuracy ranged from 1.3% to 5.71% and from −9.07% to −0.53%, respectively. The inter-day precision and accuracy ranged from 3.21% to 7.55% and from −12.6% to 0.3%, respectively. These results suggested that this method has good accuracy, precision, and reproducibility. Table 4 Matrix effect and recovery of teicoplanin in human plasma (n = 5). Nominal concentration (ug/mL)
3 20 40 IS (Sulfamethoxazole) 0.5 C.V., coefficient of variation.
Matrix effect (%)
Recovery (%)
Mean
C.V. (%)
Mean
C.V. (%)
111.27 104.50 108.10
7.63 8.17 3.57
95.46 97.67 99.83
5.65 2.87 0.75
100.52
2.06
92.28
3.77
Several chromatographic methods have been published for teicoplanin TDM as an alternative to immunological assays in clinical practice. Better correlation between immunological and chromatographic methods was noted when measuring the sum of all teicoplanin components rather than only the main components [8,16]. Therefore, we quantified the total concentration of the six major components (A3-1 and A2-1–A2-5) in patient plasma samples using this novel direct injection LC–MS/MS method. The results showed that the total concentration calculated as the sum of six components was at least 50% higher than the main components (A2-2 and A2-3) in patient samples as teicoplanin subcomponents (A2-4 and A2-5) contributed nearly half of the total concentration (data not shown), which is in agreement with the results reported by Tsai et al. [13]. Accumulation of teicoplanin components A2-4 and A2-5 may occur in human plasma following repeated administration of teicoplanin due to their higher lipophilicities [8]. Therefore, measurement of teicoplanin subcomponents (A2-4 and A2-5) is necessary to accurately quantify teicoplanin in patient plasma.
Table 5 Stability of teicoplanin at three QC levels (n = 3). Teicoplanin (A3-1 and A2-1–A5)
Concentration (g/mL)
R.E. (%)
C.V. (%)
Added
Calculated
Short-term : Exposure at RT for 6 h
3.00 20.00 40.00
3.06 20.35 39.98
2.11 1.77 −0.04
5.42 2.85 0.21
Long-term : Storage at −70 ◦ C for 30 days
3.00 20.00 40.00
2.92 19.61 38.76
−2.67 −1.97 −3.09
5.36 3.81 2.91
Freeze and thaw for 3 cycles
3.00 20.00 40.00
3.09 20.24 38.58
3.00 1.18 −3.54
4.36 7.55 2.56
RT, room temperature. R.E., relative error. C.V., coefficient of variation.
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Fig. 5. Total ion chromatograms (TIC) demonstrating (A) the similar peak shapes and RT of teicoplanin components and the IS at injection times above 1000 and (B) that the pressure of the HS-C18 column is constant.
4. Conclusion The described LC–MS/MS method enabled a simple, accurate, and robust assay for the simultaneous determination of six components of teicoplanin in human plasma. Only 20 L of plasma was required for sample preparation. In addition, it was found that the measurement of the level of subcomponents (A2-4 and A25) should be included to more accurately determine teicoplanin concentration in patient samples. This method was successfully applied in clinical practice to routine TDM for patients who received teicoplanin treatment. Acknowledgements This study was supported by a grant of the Korea Healthcare technology R&D Project, Ministry of Health & Welfare, Republic of Korea (HI14C1062). References [1] A. Borghi, C. Coronelli, L. Faniuolo, G. Allievi, R. Pallanza, G.G. Gallo, Teichomycins, new antibiotics from Actinoplanes teichomyceticus nov. sp. IV. Separation and characterization of the components of teichomycin (teicoplanin), J. Antibiot. 37 (1984) 615–620. [2] A.P. Wilson, R.N. Gruneberg, H. Neu, A critical review of the dosage of teicoplanin in Europe and the USA, Int. J. Antimicrob. Agents 4 (Suppl. 1) (1994) 1–30. [3] A. Bernareggi, A. Borghi, M. Borgonovi, L. Cavenaghi, P. Ferrari, K. Vekey, M. Zanol, L.F. Zerilli, Teicoplanin metabolism in humans, Antimicrob. Agents Chemother. 36 (1992) 1744–1749. [4] S.H. Mastin, R.L. Buck, P.A. Mueggler, Performance of a fluorescence polarization immunoassay for teicoplanin in serum, Diagn. Microbiol. Infect. Dis. 16 (1993) 17–24. [5] W.M. Awni, W.L. St Peter, D.R. Guay, M.T. Kenny, G.R. Matzke, Teicoplanin measurement in patients with renal failure: comparison of fluorescence polarization immunoassay, microbiological assay, and high-performance liquid chromatographic assay, Ther. Drug Monit. 13 (1991) 511–517.
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Journal of Chromatography B journal homepage: www.elsevier.com/locate/chromb
Direct injection LC–MS/MS method for the determination of teicoplanin in human plasma Kwang-Youl Kim a,1 , Sang-Heon Cho a,1 , Yong-Hyun Song a , Moon-Suk Nam a,b , Cheol-Woo Kim a,b,∗ a b
Department of Clinical Pharmacology, Inha University Hospital, Inha University School of Medicine, Incheon, South Korea Department of Internal Medicine, Inha University Hospital, Inha University School of Medicine, Incheon, South Korea
a r t i c l e
i n f o
Article history: Received 3 September 2015 Received in revised form 19 November 2015 Accepted 20 November 2015 Available online 24 November 2015 Keywords: Teicoplanin Multiple reaction monitoring Direct injection Therapeutic drug monitoring
a b s t r a c t A direct injection-based, simple, accurate, and robust LC–MS/MS method was developed and validated for the determination of teicoplanin in human plasma. Patient plasma samples were diluted in an aqueous buffer prior to injection into the LC–MS/MS system. Chromatographic separation was achieved using a Cadenza HS-C18 column and a gradient mixture of acetonitrile–water (both containing 0.1% formic acid) as the mobile phase at a flow rate of 0.5 mL/min. The analytes were detected in multiple reaction monitoring mode with positive ion electrospray ionization. The concentration of teicoplanin was determined as the sum of six components (A3-1, A2-1, A2-2, A2-3, A2-4, and A2-5). The calibration curve was linear over a concentration range of 1–50 mg/L, which covered the clinically accepted trough and therapeutic plasma levels. The intra- and inter-day precision and accuracy values were both less than 15%. This validated method was successfully applied to therapeutic drug monitoring of teicoplanin in routine clinical practice. Thus, we expect it to be useful for the determination of teicoplanin concentration in human plasma. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Teicoplanin is a glycopeptide antibiotics produced by Actinoplanes teichomyceticus and is commonly used to treat infections caused by gram-positive bacteria including methicillin-resistant Staphylococcus aureus (MRSA) and Enterococcus faecalis [1,2]. It comprises five major components (A2-1, A2-2, A2-3, A2-4, and A25), one hydrolysis component (A3-1), and four minor components (RS-1–RS-4). The main component is an A2 mixture that forms about 90–95% of teicoplanin. The A2-2 component is the most active compound and constitutes over 50% of the A2 mixture [3]. The concentration of teicoplanin in plasma and serum is generally determined through fluorescence polarization immunoassay (FPIA) and HPLC analysis. However, both FPIA and HPLC methods are often time consuming and cumbersome for sample pretreatment or analysis procedures [4–8]. In clinical laboratories, liquid chromatography–tandem mass spectrometry (LC–MS/MS)
∗ Corresponding author at: Department of Clinical Pharmacology, Inha University Hospital, Inha University School of Medicine, Incheon, South Korea. Fax: +82 32 882 6578. E-mail address: [email protected] (C.-W. Kim). 1 These authors contributed equally on this paper. http://dx.doi.org/10.1016/j.jchromb.2015.11.037 1570-0232/© 2015 Elsevier B.V. All rights reserved.
has become a popular assay to measure the levels of drugs, metabolites, peptides, and proteins of interest in complex biological samples owing to its excellent sensitivity and selectivity [9–11]. Recently, several LC–MS/MS methods have been reported for the quantification of teicoplanin in plasma samples. Fung et al. developed an LC–MS/MS method in which only two components (A2-2 & A2-3 isoforms) of teicoplanin were used to measure its concentration in plasma [12]. Tsai et al. described an UPLC–MS/MS method to simultaneously quantify the concentrations of six teicoplanin components (A3-1, A2-1–A2-5), including three antibiotics (vancomycin, daptomycin, and colistin), in human plasma [13]. Mueller et al. presented a high-resolution LC–MS method with online extraction based on turbulent flow chromatography for quantification of teicoplanin by measuring the five major components (A2-1–A2-5) [14]. With respect to sample preparation, the abovementioned methods mainly used protein precipitation with organic solvents. However, organic solvent-based protein precipitation often requires multistep procedures such as further dilution and the consumption of large amounts of solvent and the use of mixtures of several solvents for recovery and reduction of matrix effects [12–14]. Therefore, a more simple method for the preparation of plasma samples is required.
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It has recently been reported that a simple, direct-injection HPLC-based method using a novel hydrophobic/hydrophilic hybrid C18 column can effectively remove proteins in aqueous solutions [15]. Thus, our objective was to reduce the sample preparation procedure and develop an efficient LC–MS/MS method for teicoplanin quantification using a hybrid C18 column. In this manuscript, we report a validated LC–MS/MS method using direct injection for quantification of teicoplanin by measuring its six components (A3-1, A2-1–A2-5) in human plasma. This method is simple and robust, making it suitable for therapeutic drug monitoring (TDM) of teicoplanin in routine clinical practice.
Mass spectrometric detection was performed with an API 4000 mass spectrometer (MDS SCIEX, Toronto, Canada) operated in positive ion electrospray ionization (ESI) mode. The source parameters of the mass spectrometer were optimized and maintained as follows: collision activated dissociation (CAD) gas, 5; curtain gas (CUR), 20; gas 1 (nebulizer gas), 60; gas 2 (heater gas), 60; turbo ion spray (IS) voltage, 5300 V; source temperature, 450 ◦ C. Nitrogen was used as the nebulizer, auxiliary, curtain, and collision gases. Data processing was performed on Analyst 1.5.1 software (SCIEX).
2. Experimental
The analytical method for teicoplanin in human plasma was validated on the basis of the following parameters: specificity, linearity, lower limit of quantification (LLOQ), precision, accuracy, matrix effect, and sample stability.
2.1. Materials and reagents Teicoplanin standard (CAS Number 91032-26-7), internal standard (IS, sulfamethoxazole, CAS Number 723-46-6) (Fig. 1), and ACS reagent-grade formic acid were purchased from Sigma–Aldrich (St. Louis, MO, USA). HPLC-grade acetonitrile and methanol were obtained from Burdick and Jackson (Muskegon, MI, USA). Water was filtered using a Millipore Milli-Q system (Milford, MA, USA). Blank (drug-free) human plasma was obtained from the Clinical Trial Center at Inha University Hospital (Incheon, South Korea). 2.2. Preparation of standard and quality control (QC) samples Stock solutions of teicoplanin and IS were prepared in 50% methanol at concentrations of 10 mg/mL and stored at −20 ◦ C until dilution. Teicoplanin working solutions at concentrations of 10, 20, 50, 100, 250, and 500 g/mL were prepared via serial dilution with water. Sulfamethoxazole was diluted with 0.1% formic acid in water to prepare a stock solution at a final concentration of 500 ng/mL. Quality control (QC) working solutions were prepared at 30, 200, and 400 g/mL. All standard and quality control working solutions were kept at 4 ◦ C before analysis. Drug-free blank plasma was spiked with teicoplanin working solutions to prepare a calibration curve of 1, 2, 5, 10, 25, and 50 g/mL. In addition, QC samples for teicoplanin were prepared in drug-free plasma at concentrations of 3 g/mL (QCL; low), 20 g/mL (QCM; medium), and 40 g/mL (QCH; high) in the same manner as the calibration curves. All samples were prepared daily. 2.3. Sample preparation All samples (20 L) were added to the IS (20 L, 500 ng/mL in 0.1% formic acid solution) and diluted with 200 L of water containing 0.1% formic acid. The sample mixtures were vortexed and centrifuged for 5 min at 13,000 rpm, and the supernatant (1 L) was injected on the LC–MS/MS for analysis. 2.4. Liquid chromatography and mass spectrometry Chromatographic separation was performed on an Agilent 1200 series HPLC System (Santa Rosa, CA, USA) consisting of a binary Pump (G1312A) and an autosampler (G1367B) with thermostat (G1330B) equipped with a Cadenza HS-C18 analytical column (75 mm × 3.0 mm i.d., 3 m) maintained at 40 ◦ C. The mobile phase comprised water with 0.1% formic acid (mobile phase A) and 0.1% formic acid in acetonitrile (mobile phase B). The flow rate was 0.5 mL/min with the following gradient conditions: 0–1 min, 0% B (isocratic); 1–2 min, 0–100% B (linear gradient); 2–3 min, 100% B (isocratic); 3–3.5 min, 100–0% B (linear gradient); 3.5–6.5 min, 0% B (isocratic). Total run time was 6.5 min with 1 L injection. Autosampler temperature was set at 10 ◦ C.
2.5. Method validation
2.5.1. Specificity Specificity was evaluated by analyzing six different drug-free plasma samples to investigate potential interference from coeluting endogenous compounds in the LC peak region of teicoplanin and IS. 2.5.2. Linearity and lower limit of quantification Calibration curves were constructed by plotting the peak area ratios of a combination of six teicoplanin components to the IS vs. the nominal concentration. Linearity was tested with teicoplanin concentrations of 1–50 g/mL (1, 2, 5, 10, 25, 50 g/mL). The regression parameters of slope and y-intercept and correlation coefficient values were calculated by linear least-squares regression analysis using 1/X as the weighting factor. The LLOQ was the lowest concentration on the calibration curve that could be quantified with an acceptable precision of less than 20% and accuracy within ±20%, which was evaluated using five replicate samples. 2.5.3. Precision and accuracy The intra-day accuracy and precision of the assay were determined by analyzing three QC samples (n = 5) on the same day. The inter-day accuracy and precision were evaluated by the analysis of each QC sample over five consecutive days. Precision was determined on the basis of the coefficient of variation (C.V. (%)), and the accuracy was calculated as relative error (R.E. (%)). The acceptance criteria for precision and accuracy should be within ±15% compared to the nominal values. 2.5.4. Matrix effect and recovery The matrix effect was evaluated (n = 5) by comparing the peak areas of teicoplanin solutions spiked with diluted blank plasma samples and the same neat solutions as the three QC samples. The recovery was measured (n = 5) by comparing the peak areas of three QC samples and teicoplanin neat solution at these levels. The matrix effect and recovery of the IS was also measured using the same method. 2.5.5. Stability The stability test was performed (n = 3) with three QC samples under the following conditions: short-term and long-term temperature stability and freeze and thaw stability. 2.6. Analysis of patient samples For the determination of teicoplanin concentration in human plasma, all patient samples were centrifuged at 3000 rpm at 10 ◦ C for 10 min and stored at −70 ◦ C until analysis. The samples
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Fig. 1. Chemical structures of the major components (A2-1–A2-5) of teicoplanin and the internal standard (sulfamethoxazole).
Table 1 Optimized MS/MS conditions of six teicoplanin components and the internal standard.
Teicoplanin A2-1 Teicoplanin A2-2 & A2-3 Teicoplanin A2-4 & A2-5 Teicoplanin A3-1 Sulfamethoxazole (IS)
MRM transitions
DP (V)
CE (V)
CXP (V)
EP (V)
939.7/314.2 940.7/316.2 947.8/330.2 782.4/203.7 254.2/92.0
60 60 60 60 60
15 16 17 15 25
10 10 16 8 12
10 10 10 10 10
MRM, multiple reaction monitoring; DP, declustering potential; CE, collision energy; CXP, collision cell potential energy; EP, entrance potential.
were prepared using the above dilution method and subjected to LC–MS/MS analysis. 3. Results and discussion 3.1. Mass spectrometry We investigated the m/z values of the precursor ions for six components of teicoplanin and the IS in positive ion ESI mode through direct infusion and estimated their product ions. As a result, the doubly charged molecular ions [M + 2H]2+ of the teicoplanin components were predominantly detected in the acidic aqueous mobile phase due to the structure of the polypeptide moiety. The precursor ions of teicoplanin A3-1, A2-1, A2-2 and A2-3 (isomer), and A2-4 and A2-5 (isomer) were the doubly protonated molecular ions [M + 2H]2+ at m/z 782.4, 939.7, 940.7, and 947.8, respectively (Fig. 2). The MS/MS spectra for the six components were obtained with ramped collision energy. The product ions of teicoplanin A31, A2-1, A2-2 and A2-3 (isomer), and A2-4 and A2-5 (isomer) were abundantly produced at m/z 204.1, 314.2, 316.2, and 330.2, respectively (Fig. 2). The IS produced a protonated precursor ion [M + H]+ at m/z 254.2 and a major product ion at m/z 92.0. These major product ions were selected for multiple reaction monitoring (MRM) analysis, for which the dwell time was set to 50 ms. Detailed compound parameters are summarized in Table 1.
3.2. Liquid chromatography To obtain good chromatographic separation for teicoplanin components and the IS, we investigated mobile phase compositions with water, acetonitrile, methanol, and various pH conditions. Acetonitrile was chosen as the organic solvent as it resulted in appropriate sensitivity and peak shape of the teicoplanin components. Good ionization and fragmentation of the teicoplanin components was achieved in the presence of 0.1% formic acid. Therefore, 0.1% formic acid was included in both water and acetonitrile as mobile phase A and B, respectively. The separation of teicoplanin components and the IS was conducted with a gradient solvent system using a Cadenza HS-C18 column with dimensions of 75 mm × 3.0 mm i.d. and 3 m particle size. The initial composition was 0% solvent B for 1 min, increased to 100% solvent B over 2 min, maintained at 100% solvent B until 3 min, decreased to 0% solvent B at 3.5 min, and maintained at 0% solvent B until the end of the run time at 6.5 min. A representative chromatogram showing typical peak shapes for each analyte is depicted in Fig. 3B. Retention times for teicoplanin A3-1, A2-1, A2-2 and A2-3 (isomer), A2-4 and A2-5 (isomer), and the IS were 3.47, 3.47, 3.48, 3.49, and 3.54 min, respectively. All teicoplanin components including the IS were detected with little overlap after 3.0 min. There was no crosstalk with a dwell time of 50 ms between the MRM channel arising from the IS. Therefore, analysis is relatively quick (within 6.5 min) and reproducible without the need for further chromatographic separation of teicoplanin components. It is desirable to use a stable isotope-labeled analog of the analyte molecule as the internal standard in a quantitative MRM-MS assay. However, such compounds for teicoplanin are not commercially available. To select the proper IS on the basis of a similar retention time and matrix effect for teicoplanin components, other drugs were injected into our LC–MS/MS system. As a result, the sulfamethoxazole was selected as the most proper IS in this study because of its similar retention time (3.54 min, Fig. 3B) and matrix effect (100.52 ± 2.06%, Table 4) and the stability of ionization.
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Fig. 2. Collision-induced dissociation mass spectrum of each teicoplanin component and the IS in positive ion electrospray ionization mode. Product-ion spectrum from (a) A2-1, (b) A2-2 and A2-3, (c) A2-4 and A2-5, and (d) A3-1 obtained by fragmentation of [M + 2H]2+ at each m/z and (e) IS [M + H]+ at m/z 254.2 using a collision potential of 20 eV. The MRM transitions of the teicoplanin component and IS that produced the highest product-ion (P+ ) signals were used for teicoplanin quantification.
Table 2 Linearity data for the analysis of teicoplanin in plasma (n = 5). Nominal concentration (g/mL)
Calculated concentration (g/mL)
R.E. (%)
C.V. (%)
R2
1 2 5 10 25 50
0.96 1.97 5.05 10.36 25.60 49.08
3.70 1.30 0.92 −3.56 −2.40 1.84
2.63 2.01 3.45 3.94 1.89 1.01
0.99
R.E., relative error. C.V., coefficient of variation. R2 , coefficient of determination.
3.3. Sample preparation Direct injection is a cost-effective and time-saving method of sample preparation and can also decrease the error associated with sample handling, such as pipetting and labeling. However, the biggest drawback for direct injection of plasma samples is that con-
ventional HPLC columns are easily clogged due to the abundance of proteins such as albumin. Recently, analytical columns have been designed to exclude proteins while allowing smaller molecules to interact with unique stationary phases [15]. The Cadenza HS-C18 column has a hybrid ODS stationary phase with hydrophobic and hydrophilic groups, which helps to separate proteins and drugs of interest from biological samples in an aqueous mobile phase. We first investigated the degree to which various polar and nonpolar analytes, including teicoplanin, vancomycin, and other small molecule drugs, are separated from proteins under the above conditions. This revealed that some hydrophobic analytes such as teicoplanin components were well separated from proteins (Fig. 4A). However, some hydrophilic analytes such as vancomycin were too polar to be sufficiently separated from the larger proteins such as albumin (>30 kDa). Therefore, these polar compounds are not suitable for direct injection as they were eluted within two minutes (Fig. 4A). This column provides excellent separation for low molecular weight analytes, especially, hydrophobic compounds, from the dilute plasma proteins. Therefore, we have selected the Cadenza HS-C18 column as the direct
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Fig. 3. Representative MRM chromatograms for six components of teicoplanin and the IS in (A) blank human plasma and (B) 1 g/mL (LLOQ) teicoplanin spiked human plasma.
Fig. 4. (A) Representative chromatograms of nine drugs as standards in buffer solvent. (B) Chromatogram of the separation of diluted plasma proteins using the Cadenza HS-C18 column. The UV/VIS detector was operated in dual wavelength mode at 210 nm and 254 nm. Larger proteins were excluded from the column under these conditions (column, Cadenza HS-C18 (75 mm × 3.0 mm i.d., 3 m); flow rate, 0.5 mL/min; injection volume, 1 L; mobile phase, water/acetonitrile, both containing 0.1% formic acid, under gradient conditions).
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Table 3 Precision and accuracy for the determination of teicoplanin in human plasma (n = 5). Nominal concentration (g/mL)
3 20 40
Intra-day
Inter-day
R.E. (%)
C.V. (%)
R.E. (%)
C.V. (%)
−9.07 −2.24 −0.53
5.71 2.82 1.30
−12.60 −4.80 0.30
7.55 8.42 3.21
R.E., relative error. C.V., coefficient of variation.
injection column for teicoplanin quantification in human plasma samples. We also evaluated the effect of a dilution buffer with various acidic additives, such as formic acid and ammonium formate, while maintaining the minimum sensitivity of teicoplanin through plasma levels of <10 mg/L and minimizing the matrix effects. When plasma samples were diluted 10-fold with deionized water containing 0.1% formic acid, the sensitivity and reproducibility were suitable for the TDM analysis of teicoplanin. Finally, a post-column switching valve was employed to divert the flow from 0 to 3 min to the waste in order to minimize contamination from the eluted plasma proteins in the mass analyzer. The flow was then switched back to the MS detector from 3 to 5 min (Fig. 4B).
3.4.4. Matrix effect and recovery Matrix effect and recovery were investigated at three QC levels, as described above, and the results are summarized in Table 4. These results indicated that ion suppression or enhancement from the plasma matrix was not observed.
3.4.5. Stability The stability results showed that teicoplanin spiked in human plasma was stable for 6 h at room temperature, for 30 days at −70 ◦ C, and during three freeze–thaw cycles. The results of the stability experiments are listed in Table 5. These results demonstrated that teicoplanin is stable under the described conditions.
3.5. Robustness of direct injection methodology The robustness of our direct injection approach was investigated by sequential injections of 1000 diluted plasma samples. Reproducibility (MS sensitivity, peak shape, and retention time) was shown to be constant and column pressure was less than 6% RSD (Fig. 5). Therefore, this direct injection method has potential clinical applications for high-throughput analysis, especially, for hydrophobic analytes.
3.4. Method validation 3.6. Application 3.4.1. Specificity No interference peaks from the endogenous matrix sample were observed with peaks of the teicoplanin components and the IS (Fig. 3A). Blank plasma samples spiked with teicoplanin at the LLOQ and IS showed typical chromatograms (Fig. 3B). 3.4.2. Linearity and lower limit of quantification The calibration curves ranging from 1–50 g/mL for the sum of all six teicoplanin components in human plasma were performed on five consecutive days and the back-calculated values for each level were measured. As shown in Table 2, the calibration curves of the sum of all six teicoplanin components showed good linearity over the analysis range, with the observed deviation being within ±15% and coefficient of determination (r2 ) above 0.99 for all calibration concentrations. The response at the LLOQ was 10 times greater than the response of blank plasma. Moreover, the values of R.E. and C.V. (n = 5) for LLOQ were 3.7% and 2.63%, respectively, demonstrating that this method is sensitive enough to determine the level of teicoplanin in plasma. 3.4.3. Precision and accuracy As shown in Table 3, the intra-day precision and accuracy ranged from 1.3% to 5.71% and from −9.07% to −0.53%, respectively. The inter-day precision and accuracy ranged from 3.21% to 7.55% and from −12.6% to 0.3%, respectively. These results suggested that this method has good accuracy, precision, and reproducibility. Table 4 Matrix effect and recovery of teicoplanin in human plasma (n = 5). Nominal concentration (ug/mL)
3 20 40 IS (Sulfamethoxazole) 0.5 C.V., coefficient of variation.
Matrix effect (%)
Recovery (%)
Mean
C.V. (%)
Mean
C.V. (%)
111.27 104.50 108.10
7.63 8.17 3.57
95.46 97.67 99.83
5.65 2.87 0.75
100.52
2.06
92.28
3.77
Several chromatographic methods have been published for teicoplanin TDM as an alternative to immunological assays in clinical practice. Better correlation between immunological and chromatographic methods was noted when measuring the sum of all teicoplanin components rather than only the main components [8,16]. Therefore, we quantified the total concentration of the six major components (A3-1 and A2-1–A2-5) in patient plasma samples using this novel direct injection LC–MS/MS method. The results showed that the total concentration calculated as the sum of six components was at least 50% higher than the main components (A2-2 and A2-3) in patient samples as teicoplanin subcomponents (A2-4 and A2-5) contributed nearly half of the total concentration (data not shown), which is in agreement with the results reported by Tsai et al. [13]. Accumulation of teicoplanin components A2-4 and A2-5 may occur in human plasma following repeated administration of teicoplanin due to their higher lipophilicities [8]. Therefore, measurement of teicoplanin subcomponents (A2-4 and A2-5) is necessary to accurately quantify teicoplanin in patient plasma.
Table 5 Stability of teicoplanin at three QC levels (n = 3). Teicoplanin (A3-1 and A2-1–A5)
Concentration (g/mL)
R.E. (%)
C.V. (%)
Added
Calculated
Short-term : Exposure at RT for 6 h
3.00 20.00 40.00
3.06 20.35 39.98
2.11 1.77 −0.04
5.42 2.85 0.21
Long-term : Storage at −70 ◦ C for 30 days
3.00 20.00 40.00
2.92 19.61 38.76
−2.67 −1.97 −3.09
5.36 3.81 2.91
Freeze and thaw for 3 cycles
3.00 20.00 40.00
3.09 20.24 38.58
3.00 1.18 −3.54
4.36 7.55 2.56
RT, room temperature. R.E., relative error. C.V., coefficient of variation.
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Fig. 5. Total ion chromatograms (TIC) demonstrating (A) the similar peak shapes and RT of teicoplanin components and the IS at injection times above 1000 and (B) that the pressure of the HS-C18 column is constant.
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