2022 年 45 巻 4 号 p. 421-428
It is important to select appropriate antibiotics for infection control. Linezolid and tedizolid are newly developed and synthesized oxazolidinone antibacterial agents. It has been pointed out that there is a relationship between a high plasma concentration of the target drug and incidence of adverse effects, although it has been reported that neither linezolid nor tedizolid requires dose adjustment according to renal function. Due to the high incidence of adverse effects, both are often switched. Precise plasma concentration control by therapeutic drug monitoring (TDM) is desirable for reducing the adverse effects of both drugs and obtaining a better therapeutic effect. In this study, we aimed to establish a method for simultaneous quantification of linezolid and tedizolid in human plasma using LC coupled with tandem mass spectrometry. Sample preparation was performed by a simple operation with acetonitrile. Linezolid and tedizolid were separated by an octadecylsilyl column using a gradient elution of acetonitrile in aqueous 0.1% formic acid solution and were detected in the positive ion electrospray mode with multiple reaction monitoring. Quantification of linezolid and tedizolid ranged from 0.5 to 50 and 0.5 to 20 µg/mL, respectively. The intra-day and inter-day precision and accuracy of data were assessed and found to be acceptable. The developed method was successfully applied to measurement of the concentrations of linezolid and tedizolid. This simple method, which can simultaneously quantify both drug concentrations for daily TDM, could contribute to safer treatment of patients.
A continuous and progressive increase in bacterial resistance has resulted in an urgent need for new effective and safe antibiotics or antibacterial agents with a completely new mechanism of action that can effectively inhibit infections caused by drug-resistant bacteria. Methicillin-resistant Staphylococcus aureus (MRSA) is a multidrug-resistant pathogen that causes nosocomial infections globally.1)
Linezolid and tedizolid (Fig. 1) are newly developed and synthesized oxazolidinone antibacterial agents.2,3) Tedizolid phosphate is a prodrug that is converted to tedizolid, an active form, by phosphatase.4) Their mechanism of action is different from that of conventional protein synthesis inhibitors, which suppress the translation initiation reaction in protein synthesis by binding to the 50s subunit of the bacterial ribosome and inhibiting the formation of an initiation complex and inhibiting protein synthesis.2,4) The applicable bacterial species is MRSA, and the indications are deep skin infections, chronic pyoderma, secondary infections such as external phase, burns and surgical wounds.2,4,5)
Bone marrow suppression such as thrombocytopenia and anemia is known as a typical adverse event of oxazolidinone antibacterial agents.5–7) Linezolid causes a high incidence of thrombocytopenia and it has been reported that the incidence of thrombocytopenia caused by linezolid is higher in patients with impaired renal function and in patients who receive long-term administration of linezolid for 14 d or more.8,9) Due to the high incidence of thrombocytopenia caused by linezolid, it is switched to other agents such as tedizolid, which has been reported to cause a lower incidence of thrombocytopenia than that caused by linezolid.6,10,11) Since the plasma trough concentration of linezolid correlates with the incidence of thrombocytopenia and inadequate therapeutic efficacy, maintenance of a trough concentration between about 3.6 to 8.2 µg/mL by therapeutic drug monitoring (TDM) has been reported to be helpful for continuous treatment.12) The linezolid dose needed to achieve a pharmacokinetic/pharmacodynamic (PK/PD) target has been reported to be associated with the area under the plasma drug concentration–time curve (AUC24)/minimum inhibitory concentration (MIC) ratio ≥100.13,14) Matsumoto et al. reported that the efficacy could be guaranteed if the AUC of linezolid was 200 µg·h/mL or more from the MIC value of MRSA isolated from the infected site. The recommended trough concentration of linezolid would be needed to achieve AUC 200 µg·h/mL or more.12) Tedizolid does not require dose adjustment even in patients with hepatic or renal dysfunction. However, it was reported that the area under the curve of tedizolid was correlated with body weight of patients.15) Those reports suggested that TDM of tedizolid is useful for pharmacological treatment with few adverse effects. Several methods for quantification of plasma linezolid have been reported,16–19) there have been only a few methods reported for quantification of plasma tedizolid.19–21)
The aim in this study was to develop an easier method for simultaneous determination of linezolid and tedizolid in human plasma using LC with tandem mass spectrometry (LC/MS/MS) and to validate the method for appropriate use of antibacterial agents and safe drug treatment.
Linezolid (MW 337.35, CAS# 165800-03-3) powder (purity, 99.78%) was purchased from MedChemExpress U.S.A. (NJ, U.S.A.). Tedizolid (MW 370.34, CAS# 856866-72-3) powder (purity, ≥98%) and linezolid-d3 (MW 340.36, CAS# 1127120-38-0) powder (purity, 98.0%) were purchased from LKT Laboratories, Inc. (MI, U.S.A.) and Toronto Research Chemicals (ON, Canada), respectively. Acetonitrile, methanol, dimethyl surfoxide (DMSO) and formic acid were purchased from FUJIFILM Wako Pure Chemical Corp. (Osaka, Japan). All of the reagents were of the highest grade available and used without further purification.
Standard Solutions, Calibration Standards and Quality Control (QC) SamplesStock solutions (each 1.0 mg/mL) of linezolid and tedizolid used to make calibration standards and QC samples were prepared by dissolving 5 mg of each compound in 5 mL water and DMSO. The stock solutions were further diluted with 50% methanol to obtain working solutions at several concentrations. Calibration standards and QC samples in plasma were prepared by diluting the corresponding working solutions with blank human plasma. Final concentrations of calibration standards were 0.5, 2.0, 4.0, 8.0, 16, 28, 40 and 50 µg/mL for linezolid and 0.5, 2.0, 4.0, 6.0, 8.0, 12, 16 and 20 µg/mL for tedizolid. The final concentrations of QC samples for linezolid and tedizolid in plasma were 0.5, 1.0, 20 and 40 µg/mL, 0.5, 1.0 10 and 16 µg/mL, respectively. Linezolid-d3 as an internal standard (IS) stock solution was made at an initial concentration of 1.0 mg/mL in methanol and stored at 4 °C (Pharmaceutical refrigerator MPR-414F-PJ, Panasonic Corp., Osaka, Japan). The IS working solution (10 µg/mL) was made from the stock solution using methanol for dilution. Other stock solutions were stored at −20 °C (biomedical freezer MDF-U539, SANYO Electric Co., Ltd., Osaka, Japan). The refrigerator and freezer alarms were set to ±5 and ±10 °C, respectively. During the study, we monitored the temperature of the refrigerator and freezer once a week. The temperature range was 4 ± 2 and −20 ± 4 °C, respectively. The alarm had never sounded due to such as the temperature deviance.
Sample PreparationFresh frozen plasma, leukocytes reduced, Nisseki 240 (FFP-LR240) as blank human plasma was purchased from Japanese Red Cross Society (Lot Nos. A-02-0120-1849, A-02-0623-3271, A-02-0623-6963, A-02-0120-1844, A-02-2120-3298 and A-02-0321-1986). Sodium hydrate citrate was used for anti-coagulation in this human plasma.
Before analysis, the plasma sample was thawed to room temperature. In a 2.0 mL tube, a solution of 40 µL each of linezolid and tedizolid (for standard and QC samples) or 80 µL of 50% methanol (sample) was added to 320 µL of plasma. One hundred microliters of IS solution (10 µg/mL) and 1000 µL of acetonitrile were added to the mixture and the mixture was shaken vigorously for 1 min. After centrifugation at 16600 × g for 15 min at 4 °C, the supernatant was taken and diluted 2-fold with water for injection. The diluted solution was further centrifuged at 16600 × g for 3 min at 4 °C. The solution was injected into an LC-MS/MS system.
LC/MS/MS AnalysisLC was performed on a Shimadzu Prominence 20 A System (Shimadzu, Kyoto, Japan) with a TSKgel® ODS-100 V 5 µm column (2.0 × 50 mm, particle size of 5 µm, #0021457, TOSOH Corp., Tokyo, Japan) and inline guard column (TSKgel guardgel ODS-100 V 5 µm, 2.0 × 10 mm, #0021841 and #0019308, TOSOH Corp.). A gradient program was employed with the mobile phase, combining solvent A (0.1% formic acid in water) and solvent B (acetonitrile) as follows: 20% B (0–0.01 min), 20–95% B (0.01-0.60 min), 95% B (0.60–3.00 min), 95–20% B (3.00–3.20 min), and 20% B (3.20–6.00 min). The flow rate was 0.2 mL/min (20% B) and the injection volume was 1 µL. The column and sample temperatures were maintained at 40 and 4 °C, respectively.
Positive ion electrospray (ESI)-MS/MS analysis was performed using an API3200™ LC/MS/MS system with multiple reaction monitoring (MRM) (Applied Biosystems, Foster City, CA, U.S.A.). MRM was performed by monitoring the transitions summarized in Table 1. Parameter settings were as follows: source temperature of 700 °C, ion-spray cone voltage of 5500 V, curtain gas of 10 psi, ion source gas 1 of 70 psi, ion source gas 2 of 50 psi, and collision gas of 10 arbitrary units. Data were acquired and analyzed using Analyst® software version 1.6.3. (Applied Biosystems).22)
| Analyte | Precursor ion (m/z) | Product ion (m/z) | Dwell time (ms) | DP (V) | EP (V) | CE (V) | CEP (V) | CXP (V) |
|---|---|---|---|---|---|---|---|---|
| Linezolid | 338.163 | 56.000 | 333.30 | 41.0 | 8.0 | 65.0 | 16.0 | 2.0 |
| Tedizolid | 371.030 | 343.200 | 333.30 | 56.0 | 6.5 | 21.0 | 24.0 | 4.0 |
| Linezolid-d3 (IS) | 341.161 | 57.200 | 333.30 | 51.0 | 6.5 | 65.0 | 14.0 | 0 |
DP, Declustering potential; EP, Entrance potential; CE, Collision energy; CEP, Collision cell entrance potential; CXP, Collision cell exit potential.
The present method was fully validated in accordance with United States Food and Drug Administration, Guidance for Industry: Bioanalytical Method Validation and European Medicines Agency, Guideline on Bioanalytical Method Validation.23,24) Pooled blank human plasma was obtained as described above and used for method validation including calibration curve, selectivity, intra-day and inter-day precision and accuracy, recovery, matrix effect and stability.
Linearity of Calibration CurvesCalibration curves were constructed using stock solutions in blank plasma. The samples were pretreated as described above and analyzed. Calibration curves were constructed by plotting the peak ratio (standard to internal standard) versus the nominal concentration. These calibration curves were composed of a standard sample for a calibration curve having 8 concentrations including a blank sample, a zero sample and a low limit of quantification (LLOQ) sample. Analytical response was calculated as the ratio of the peak area of each analyte to that of IS, and weighted linear regression (1/x) of actual concentration versus analytical response was conducted for each analytical batch according to previous reports.19,20,22) The regression equation of the calibration curve was calculated using points other than the zero point.
Precision, Accuracy, LLOQ and RecoveryIntra-day precision and accuracy were assessed by analyzing six replicates at four different concentrations on the same day. Inter-day precision and accuracy were assessed by analyzing the replicates at four different concentrations on 6 different days. The replicates were prepared and analyzed. The precision was obtained as the relative standard deviation (RSD). The accuracy was expressed as relative error (RE). RE (%) was calculated as [(found concentration−theoretical concentration)/theoretical concentration×100]. The acceptable limit for accuracy and precision was ≤ ± 15% except for LLOQ, for which the acceptable limit was ≤ ± 20%. LLOQ was defined as the concentration with a signal-to-noise (S/N) ratio of at least 10 and precision and accuracy data.
Recovery was assessed by spiking known amounts of linezolid and tedizolid into blank plasma and comparing the peak areas of analytes spiked after sample preparation that represent 100% recovery.
Matrix EffectsAs described in the former section, human blank plasma samples were purchased. For measurement of linezolid and tedizolid, the matrix effect was assessed by measuring the peak area in the presence of a matrix (peak area of the analyte spiked after sample preparation) and the peak area in the absence of a matrix (peak area of an equivalent amount of analyte prepared in 50% methanol). The matrix effect was calculated using the following equation:
 |
Carry-over was assessed by injections of a highest calibration sample (20 µg/mL) and a zero sample. The area responses of the blank samples were compared to the mean area response of the LLOQ. The peak area of the blank samples following the highest calibration should not exceed 20% of the peak area of the LLOQ.
StabilityThe stability of linezolid and tedizolid in plasma was investigated. The short-term stabilities were assessed after storing linezolid and tedizolid for 2 weeks and 4 weeks in plasma at 4 and −20 °C, respectively. The long-term term stability was assessed after storing linezolid and tedizolid for 8 weeks in plasma at room temperature.
The stability of linezolid and tedizolid in stock solutions was also evaluated. The long-term stability of linezolid and tedizolid in stock solutions after 12 weeks of storage at 4 and −20 °C was assessed. Freeze–thaw stability of the stock solution was also assessed after storage at −20 °C for 12 weeks and three freeze–thaw cycles (−30 °C to room temperature).
In the first part of this study, ESI-MS/MS (positive mode) was used for detecting linezolid, tedizolid and linezolid-d3 in the MS tuning. Table 1 shows the ion pairs selected for MRM and parameter settings. The product ions of linezolid, tedizolid and linezolid-d3 were identified by MRM analysis and were determined with consideration of the sensitivity, selectivity and interference to other peaks. Although we selected a single transition for quantification of each analyte, no interfering peak was observed in any of the tested lots of blank human plasma samples.
Sample PretreatmentConditions for sample preparation were then developed. Linezolid was dissolved in water to prepare 1 mg/mL stock solution. Tedizolid was prepared in the same manner using DMSO since tedizolid has low solubility in water (about 0.1 mg/mL). Yu et al. reported that they prepared a stock solution of 1.0 mg/mL methanol solution, but tedizolid did not dissolve in our study.20) During the study, when the stock solutions of linezolid and tedizolid were diluted with water, linezolid and tedizolid adsorbed onto the surface of tube in the low concentration range containing LLOQ. This adsorption could be suppressed to some extent by diluting the stock solutions with 50% methanol. We decided to dilute the stock solutions of both linezolid and tedizolid with 50% methanol in subsequent studies. In addition, it is important to select an appropriate method for extraction of linezolid and tedizolid from the matrix. Tanaka et al. used a solid-phase extraction system to extract linezolid, tedizolid and daptomycin.19) A solid-phase extraction system is convenient, but there are concerns about the high costs of such systems (about $450 (¥50000)/pk) and some work process. In this study, acetonitrile and 50% methanol were selected for deproteination from human plasma with consideration of various aspects such as daily measurement cost. Sufficient separation of linezolid and tedizolid from human plasma was not obtained with only acetonitrile although the conditions of treatment were tested referring to a previous report20) (data not shown). We succeeded to separate linezolid and tedizolid from human plasma with easy steps (Fig. 2). Iqbal pretreated plasma samples with a liquid-liquid extraction technique using ethyl acetate as an extracting reagent for quantitative measurement of tedizolid alone.21) However, it is necessary to evaporate the highly lipophilic organic solvent and to redissolve in the mobile phase for LC/MS injection. Acetonitrile was suitable for recovering linezolid, which is highly soluble in water, and tedizolid, which is poorly soluble in water.
Representative chromatographs of linezolid and tedizolid (each final concentration: 0.5 µg/mL) and linezolid-d3 (internal standard, final concentration: 1 µg/mL) in blank human plasma were obtained.
Under these conditions, the recovery of linezolid and tedizolid from human plasma was analyzed. It was shown that the recovery of both linezolid and tedizolid from plasma was about 100% (Table 2), suggesting that the present method has very little loss in the extraction process.
| Analyte | Concentration (µg/mL) | Recovery (%) | Matrix effect (%) | ||
|---|---|---|---|---|---|
| Mean ± S.D. | RSD (%) | Mean ± S.D. | RSD (%) | ||
| Linezolid | 1.0 | 103.15 ± 3.30 | 3.20 | 95.95 ± 6.88 | 7.17 |
| 40 | 99.52 ± 1.61 | 2.40 | 100.13 ± 2.17 | 2.17 | |
| Tedizolid | 1.0 | 97.96 ± 5.25 | 5.36 | 99.95 ± 1.40 | 1.47 |
| 16 | 102.96 ± 0.85 | 3.39 | 98.94 ± 3.39 | 3.42 | |
The value of recovery represents the mean ± standard deviation of 6 measurements. The value of matrix effect represents the mean ± S.D. of 5 measurements. The precision was expressed as the relative standard deviation (RSD).
Calibration standards were established by spiking at least seven different concentrations containing LLOQ of linezolid and tedizolid to blank human plasma. In previous reports, the calibration curves for linezolid had concentration ranges of 0.75–50, 0.1–100, 0.4–40, 0.02–20, 1–50 µg/mL, respectively.18,19,25–27) On the other hand, the ranges of calibration curves for tedizolid were 0.005–5 (human), 0.005–5 (rat), 0.00074–1.5 (rat) µg/mL, respectively.19–21) The LLOQ of both linezolid and tedizolid was 0.5 µg/mL (Fig. 2) in this study. The present method showed good linearity every time in both linezolid (r2 > 0.998) and tedizolid (r2 > 0.998). In the preliminary study, good linearity was also confirmed in the range of 0.5–50 µg/mL.
The intra-day precision and inter-day precision of both linezolid and tedizolid as well as the accuracy were investigated at four different concentrations. The data are summarized in Table 3. In the quantification method with human plasma, the intra-day precision of linezolid and that of tedizolid ranged from 1.68 to 5.05 and 2.51 to 5.90%, respectively. The intra-day accuracy of linezolid and that of tedizolid ranged from 0.75 to 4.67 and 0.50 to 12.00%, respectively. The inter-day precision of linezolid and that of tedizolid ranged from 3.61 to 11.41 and 3.62 to 18.94%, respectively, and the inter-day accuracy of linezolid and that of tedizolid ranged from 0 to −8.77 and 0.15 to 5.72%, respectively. The precision and accuracy are recommended to be within 15%, except for LLOQ (those of LLOQ being within 20%). These results indicate that the present method is highly reliable and has good precision and accuracy.
| Analyte | Conc. (µg/mL) | Intra-day (N = 5) | Inter-day (N = 5) | ||||
|---|---|---|---|---|---|---|---|
| Found conc. (µg/mL) | RSD (%) | RE (%) | Found conc. (µg/mL) | RSD (%) | RE (%) | ||
| Linezolid | 0.5 | 0.49 | 5.05 | −0.93 | 0.45 | 11.41 | −8.77 |
| 1.0 | 1.04 | 2.84 | 4.40 | 1.00 | 7.82 | 0.29 | |
| 20 | 20.93 | 1.93 | 4.67 | 20.00 | 4.42 | 0 | |
| 40 | 40.30 | 1.68 | 0.75 | 39.14 | 3.61 | −2.15 | |
| Tedizolid | 0.5 | 0.56 | 5.90 | 12.00 | 0.53 | 18.94 | 5.72 |
| 1.0 | 1.08 | 2.51 | 7.60 | 1.03 | 8.86 | 3.21 | |
| 10 | 10.05 | 3.48 | 0.50 | 10.02 | 3.62 | 0.15 | |
| 16 | 16.30 | 3.41 | 1.88 | 16.06 | 3.75 | 0.38 | |
The precision was expressed as the relative standard deviation (RSD). The accuracy was expressed as relative error (RE).
We then investigated the matrix effect. Deuterium-labeled linezolid was used as the internal standard for both linezolid and tedizolid (Fig. 3). The results of the matrix effect are shown in Table 2. The precision of the matrix effect is required to be 15% or less between individuals. Our results were consistent with this and suggested that the response of the component under analysis is hardly influenced by matrix-derived components in the sample.
Representative chromatographs of linezolid and tedizolid and linezolid-d3 (internal standard, final concentration: 1 µg/mL) in blank human plasma were obtained.
We also analyzed the precision and the accuracy in multiple lots of blank human plasma to assess whether the IS can compensate for variations in the matrix effect and recovery. As shown in Table 4, no significant variability among the lots was observed. During the quantification of plasma, the precision of linezolid and that of tedizolid ranged from 4.20 to 10.25 and 2.63 to 8.42%, respectively. The accuracy of linezolid and that of tedizolid ranged from 0 to −17.40 and −0.05 to −16.17%, respectively. It was shown that the human plasma used in this study could be used as blank plasma without any problem for determining the concentrations of linezolid and tedizolid.
| Analyte | Spiked conc. (µg/mL) | Found conc. (µg/mL) | RSD (%) | RE (%) | |||||
|---|---|---|---|---|---|---|---|---|---|
| Lot #1 | Lot #2 | Lot #3 | Lot #4 | Lot #5 | Lot #6 | ||||
| Linezolid | 0.5 | 0.43 | 0.42 | 0.38 | 0.42 | 0.36 | 0.47 | 10.25 | −17.40 |
| 1.0 | 0.96 | 0.95 | 1.09 | 1.14 | 0.86 | 1.00 | 10.16 | 0 | |
| 20 | 20.10 | 19.70 | 20.10 | 21.40 | 18.90 | 19.52 | 4.20 | −0.25 | |
| Tedizolid | 0.5 | 0.41 | 0.42 | 0.42 | 0.44 | 0.42 | 0.41 | 2.63 | −16.17 |
| 1.0 | 0.91 | 0.93 | 0.89 | 0.94 | 0.89 | 1.10 | 8.42 | −5.63 | |
| 10 | 10.20 | 10.40 | 9.63 | 10.32 | 9.78 | 9.66 | 3.44 | −0.05 | |
The precision was expressed as the relative standard deviation (RSD). The accuracy was expressed as relative error (RE).
Carry-over was also investigated. Carry-over is the effect of the components for analysis remaining on the analytical instrument on the low range value. In this study, the response of a blank sample after measuring the standard sample for the calibration curve at the highest concentration (20 µg/mL) must be 20% or less of the component to be analyzed at LLOQ and 5% or less of the internal standard. The carry-over of linezolid and that of tedizolid were 5.92 and 3.08%, respectively. The carry-over was 3.05% for the internal standard substance. From these results, it is considered that carry-over is almost negligible.
StabilityIn addition, the stabilities in various conditions were evaluated. The short-term stability (for 2 weeks at 4 and −20 °C) and long-term stability (for 4 weeks at 4 and −20 °C and for 8 weeks at room temperature and −20 °C) of linezolid and tedizolid in human plasma were investigated. According to the guidelines, the average accuracy at each concentration should be within 15% of the theoretical value. The data are summarized in Table 5. The accuracy of both linezolid and tedizolid of short-term storage was 0.37 to 13.33% at a low concentration (1.0 µg/mL) at 4 and −20 °C. At a high concentration (40 µg/mL of linezolid and 16 µg/mL of tedizolid), the accuracy of both linezolid and tedizolid of short-term storage was calculated to be −0.21 to 13.20% at 4 and −20 °C. On the other hand, the accuracy of both linezolid and tedizolid of long-term storage (4 and 8 weeks) was 0.13 to 14.33 and 3.00 to 6.67% at a low concentration (1.0 µg/mL) at 4 or −20 °C, respectively. At the high concentration (40 µg/mL of linezolid and 16 µg/mL of tedizolid), the accuracy of both linezolid and tedizolid of long-term storage (4 and 8 weeks) was −0.42 to 4.50 and −2.50 to 5.33%, respectively. The accuracy of linezolid and tedizolid of long-term storage (8 weeks) at room temperature was 15.83 and 3.00% at the low concentration, respectively. At the high concentration, the accuracy of linezolid and tedizolid of long-term storage at room temperature was 12.78 and −3.54%, respectively. Summarizing the stability of linezolid and tedizolid in human plasma, there was a tendency for the accuracy to be larger at a low concentration than at a high concentration. No certain rules were found regarding the storage period and storage temperature. This result showed that the degree of change in stability of linezolid and tedizolid in human plasma may not alter largely for up to 8 weeks when stored at 4 and −20 °C. In case of long-term storage of linezolid samples, it would be better to store them frozen or refrigerated.
| Analyte | Conc. (µg/mL) | 2 weeks −20 °C | 4 weeks −20 °C | 8 weeks −20 °C | 2 weeks 4 °C | 4 weeks 4 °C | 8 weeks RT | ||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| RSD (%) | RE (%) | RSD (%) | RE (%) | RSD (%) | RE (%) | RSD (%) | RE (%) | RSD (%) | RE (%) | RSD (%) | RE (%) | ||
| Linezolid | 1 | 1.47 | 13.33 | 4.12 | 0.13 | 5.89 | 3.00 | 3.65 | 12.78 | 4.61 | −1.80 | 1.90 | 15.83 |
| 40 | 1.72 | 12.20 | 1.05 | −4.50 | 0.90 | 5.33 | 1.70 | 13.20 | 5.39 | −4.17 | 2.99 | 12.78 | |
| Tedizolid | 1 | 1.44 | −1.37 | 2.38 | 11.00 | 2.17 | 6.67 | 2.46 | 0.37 | 1.82 | 14.33 | 2.57 | 3.00 |
| 16 | 3.15 | −0.63 | 4.54 | −0.42 | 2.22 | −2.50 | 2.36 | −0.21 | 3.46 | 2.71 | 5.88 | −3.54 | |
N = 3. RT, room temperature. The precision was expressed as the relative standard deviation (RSD). The accuracy was expressed as relative error (RE).
We also evaluated the stability of the solution itself. The ratios of the peak of the solution to the peak of the solution prepared on the day when stored at 4 and −20 °C for 12 weeks were calculated to be 80.18 and 85.68% for linezolid, respectively. Similarly, for tedizolid, the ratios at 4 and −20 °C were 91.67 and 93.00%, respectively. The peak percentages when stored at −20 °C for 12 weeks and frozen and thawed three times were calculated to be 93.24 and 88.49% for linezolid and tedizolid, respectively. The results suggested that the solution should be stored frozen rather than at room temperature.
Nukui et al. reported that patients with renal impairment are more likely to have a high plasma concentration of linezolid, although dose adjustment of linezolid is not necessary even in patients with hepatic or renal dysfunction.8) Linezolid is switched to tedizolid due to the development of adverse effects such as thrombocytopenia.6) As oxazolidinone antibacterial agents, the mechanisms of action of are similar, but the pharmacokinetics of the two drugs are very different.28) In order to reduce the adverse effects of both drugs and obtain a better therapeutic effect, TDM is considered to be useful, and the method presented here that enables simultaneous quantification will contribute to safer drug treatments. We apply this method to TDM and we are collecting samples from patients who received linezolid or tedizolid for safer pharmaceutical treatment. Preparations to show that the plasma concentration can be measured using this method are in progress.
In the present study, we developed a method for the quantification of linezolid and tedizolid in human plasma using LC-MS/MS. Sample preparation was conducted by a simple operation. Cost considerations are also required for routine TDM measurement in addition to method simplicity and the present method is considered useful. After full validation, the present method for measuring linezolid and tedizolid using human plasma was shown to be valid.
The authors are grateful to Dr. Ayako Furugen and Dr. Soyoko Kaburaki in Hokkaido University for giving us appropriate advice for full validation.
The authors declare no conflict of interest.
