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Original research
Therapeutic drug monitoring of linezolid: HPLC-based assays for routine quantification of linezolid in human serum and cerebrospinal fluid
  1. Stefan Günther1,2,
  2. Andreas Reimer2,
  3. Horst Vogl2,
  4. Stephan Spenke3,
  5. Hanns-Christian Dinges1,
  6. Leopold Eberhart1,
  7. Götz Geldner3
  1. 1 The Department of Anaesthesiology & Intensive Care, Philipps-Universitat Marburg, Marburg, Hessen, Germany
  2. 2 the Department of Pharmacy, Regionale Kliniken Holding RKH GmbH, Ludwigsburg, Baden-Württemberg, Germany
  3. 3 Anaesthesiology and ICM, Regionale Kliniken Holding RKH GmbH, Ludwigsburg, Baden-Württemberg, Germany
  1. Correspondence to Stefan Günther, the Department of Anaesthesiology & Intensive Care, Philipps University Marburg, Philipps-Universitat Marburg, Marburg, Hessen, Germany; stefan.guenther{at}rkh-kliniken.de

Abstract

Objective Therapeutic drug monitoring (TDM) of linezolid can prevent over- and under-dosing in critically ill patients and can be crucial to successful antibiotic treatment. Quick and simple high-performance liquid chromatography (HPLC) assays for the detection of linezolid in human serum and cerebrospinal fluid (CSF) were developed in this study.

Methods The methods used an Atlantis T3 5.0 µm stationary phase. The mobile phase A contained water (99.4% m/m) and formic acid (0.6% m/m) (pH 2.30). The mobile phase B contained acetonitrile (93.6% m/m), water (6% m/m) and formic acid (0.4% m/m). The methods were isocratic, using 23% of mobile phase B and 77% of mobile phase A. Ultraviolet absorbance detection at 252 nm was used. For sample preparation an internal standard was added, and acetonitrile/methanol was added for protein precipitation.

Results The methods were investigated for linearity, specificity, accuracy, and precision. Stability of linezolid and internal standard was assessed. The retention times of linezolid were 8.5 min and 8.1 min, and the single run time was 15 min. Linezolid was quantified from the lower limit of quantification (0.2 mg/L) to the upper limit of quantification (50 mg/L, 75 mg/L, and 100 mg/L). In routine analysis a high variability of serum and CSF levels was observed and the mean CSF/serum ratio was 0.71±0.16.

Conclusion The developed assays enable the study of correlations between the applied dosage, serum concentration and CSF concentration. Additionally, studies with a higher number of samples can be performed to investigate the penetration of linezolid into the central nervous system.

  • therapeutic drug monitoring
  • critical care
  • clinical medicine
  • drug monitoring
  • microbiology

Data availability statement

No data are available. Not applicable.

https://doi.org/10.1136/ejhpharm-2021-003036

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    Introduction

    Linezolid is an oxazolidinone derivative with a broad spectrum of activity against Gram-positive bacteria. It binds to the ribosomal 50S subunit of bacteria in the translation initiation reaction, leading to an inhibition in the early stage of protein synthesis.1 In times of an alarming resistance increase against most of the currently available antibiotics, optimisation of antibiotic dosing regimens is an important strategy for prevention.2 Therapeutic drug monitoring (TDM) is an option that is gaining a major role in optimising treatment with several antibiotics.3 Especially on intensive care units (ICUs), TDM allows accurate dosing in critically ill patients that have disturbed pharmacokinetics due to various stages of organ failure and are therefore prone to over- and under-dosing. Moreover, a high number of bloodstream infections in critically ill patients are caused by Gram-positive bacteria, including methicillin-resistant Staphylococcus aureus (MRSA). Linezolid is one antibiotic substance with good in vitro and in vivo activity against these organisms and is therefore frequently used in critically ill patients in ICUs.4 TDM of linezolid in critically ill patients is recommended by a position paper, but assays for routine TDM are sparsely implemented.5 To satisfy the need of ICUs, simple methods are described to determine linezolid in human serum and cerebrospinal fluid (CSF). The aim of this study is to demonstrate the development, validation and routine use of isocratic, internal standard high-performance liquid chromatography (HPLC) assays for linezolid in human serum and CSF.

    Materials and methods

    Antibacterial agents and other substances

    We used linezolid solution for infusion, commercially available from Dr Friedrich Eberth Arzneimittel (Ursensollen, Germany), 2-ethoxybenzamide from Sigma Aldrich (Taufkirchen, Germany), and porcine serum from bio&SELL GmbH (Feucht, Germany). Patient serum and patient CSF were received from ICUs for TDM. Dichloromethane, ethyl acetate, sodium sulfate, pentane, and ethanol were purchased from Th. Geyer GmbH & Co KG (Renningen, Germany).

    Solvents

    We purchased formic acid, sodium hydroxide, methanol (HPLC grade) and acetonitrile (HPLC grade) from Th. Geyer GmbH & Co KG (Renningen, Germany). Purified water was purchased from Fresenius Kabi Deutschland GmbH (Bad Homburg, Germany).

    High-performance liquid chromatography

    An HPLC system by Shimadzu was used that contains a temperate autosampler, column oven and ultraviolet (UV)-Vis detector. Labsolution (Shimadzu, Germany) software was used to control the chromatographic system of the internal standard-based method.

    The stationary phase was Atlantis T3 5 µm, 15 cm × 4.6 mm column (Waters Corporation, Milford, Massachusetts, USA).

    The mobile phase A contained water (99.4% m/m) and formic acid (0.6% m/m), and was adjusted to pH 2.30 by the addition of 1M sodium hydroxide. The mobile phase B contained acetonitrile (93.6% m/m), water (6% m/m) and formic acid (0.4% m/m). We used an isocratic method consisting of 23% mobile phase B and 77% mobile phase A.

    The pump flow rate was 1.0 mL/min. UV absorbance detection was used at 252 nm (linezolid) and 293 nm (2-ethoxybenzamide). The column oven temperature was set to 20°C in routine use but was also validated at 40°C. The performance of the method at 40°C can be used if a signal interferes with linezolid or the separation is incomplete.

    The method was run for 15 min, and the retention times were 8457 min for linezolid and 12 124 min for 2-ethoxybenzamide at 20°C. At 40°C the retention times were 8088 min (linezolid) and 10 773 min (2-ethoxybenzamide).

    Reference standards

    There are no commercially available chemical reference standards of linezolid. Accordingly, a characterised standard was prepared in-house using linezolid 2 mg/mL solution for infusion. The aqueous layer was extracted with Dichlormethane (DCM) and separated. The organic layer was dried over sodium sulfate, filtrated and concentrated. Recrystallization with ethanol yields a white solid with a melting point of <170°C. According to the literature, the reported melting point of linezolid is 176–178°C.6 So, the white solid was dissolved in ethyl acetate and precipitated by adding pentane. A melting point of 177–178°C was obtained and the infrared (IR) analysis report showed 96.56% correlation with a linezolid reference spectrum. This standard prepared in-house was used to quantify the content of linezolid solution for infusion that was used to prepare the standards for calibration curves.

    Sample preparation

    Samples were prepared by mixing 250 µL patient serum or CSF with 50 µL internal standard (2-ethoxybenzamide 125 mg/L) and 500 µL acetonitrile/methanol (1:1) for precipitation. The samples were mixed for 10 s and centrifuged at 9720g for 10 min; 200 µL of the supernatant was diluted with 460 µL water and 50 µL of this mixture was injected.

    Results

    Selectivity

    Selectivity of the analytical method was proven using six individual sources of the appropriate blank matrix (human serum), which were individually analysed and evaluated for interference; 16.7% of the blank matrix samples were expected to interfere with linezolid or 2-ethoxybenzamide using the method at 20°C column oven temperature. Therefore, the methods at 40°C column oven temperature were developed.

    Carry-over

    To prevent carry-over we injected blank samples after high concentration samples.7 There was no carry-over detected in the blank samples.

    Lower limit of quantification

    The lower limit of quantification (LLOQ) is defined as the lowest concentration of analyte in a sample which can reliably be quantified, with an acceptable accuracy and precision. LLOQ is aimed to be at least five times the signal of a blank sample.7 For these analytical methods the LLOQ for linezolid is 0.2 mg/L.

    Calibration curve

    The defined target serum concentration range of linezolid is 6–8 mg/L for continuous infusion due to the pharmacokinetic/pharmacodynamic index AUC0-24/minimum inhibitory concentration (MIC). Maximum efficacy of linezolid is demonstrated at an AUC0-24/MIC ratio of 80–120.8 Reported MICs for linezolid of common pathogens are 1 mg/L and 2 mg/L.9

    According to the target concentration range a minimum of six calibration concentration levels were used for each method.7 The LLOQ is defined as being the lowest calibration standard and the highest calibration standard defines the upper limit of quantification (ULOQ) as seen in table 1. LLOQ is 0.2 mg/L for all methods, the ULOQ is 50 mg/L for the methods using CSF, 75 mg/L using serum at 40°C, and 100 mg/L using serum at 20°C.

    View this table:
    Table 1

    Linezolid concentration levels used for the calibration curves. Every calibration sample contains 25 mg/L internal standard (2-ethoxybenzamide)

    For the calibration standards porcine serum and residual material of human CSF was used. To prepare the calibration standards 200 µL matrix was spiked with 50 µL linezolid solution (target concentration level ×5 mg/L). The following steps were performed to analogue the sample preparation. All calibration curves were performed using freshly spiked samples. The correlation between mean area ratio and concentration ratio was strong for all calibration curves (R2 >0.9999) as shown in figure 1.

    Figure 1
    Figure 1

    Mean area ratio plotted against the concentration ratio of linezolid and 2-ethoxybenzamide. CSF, cerebrospinal fluid.

    Accuracy

    The accuracy describes the closeness of the determined value obtained by the method to the nominal concentration of the analyte. Accuracy was assessed on samples spiked with known amounts of the analyte. These samples were spiked independently from the calibration standards and were analysed against the calibration curves. For validation of the accuracy we analysed LLOQ, low, medium and high concentration samples. The mean concentration within a value of 15% from the nominal values is commonly considered acceptable, except for the LLOQ which is acceptable within 20% of the nominal value.7 The accuracy was demonstrated with all mean concentrations between 87.9% and 103.2% of the nominal value.

    Precision

    The precision of the analytical method describes the closeness of repeated individual measures of analyte in the same sample. Precision can be expressed as the relative standard deviation (RSD). Precision of the analytical method should be demonstrated for the LLOQ, low, medium and high sample concentrations. The RSD value should not exceed 15% for the low, medium and high concentration samples, except for the LLOQ which should not exceed 20%.7 Precision was demonstrated for every method with all RSD values ranging between 0.1% and 11.3%.

    Linezolid stability

    To detect stability, we analysed the degradation of linezolid and 2-ethoxybenzamide under relevant conditions. In the stability test linezolid and 2-ethoxybenzamide were evaluated in aqueous stock solution at −32°C for long-term stability of internal standard and quality control (QC) samples. The stability of linezolid and 2-ethoxybenzamide in the analysed matrix post-preparative was also investigated for a period of 5 days at different storage conditions. Figure 2 demonstrates the observation of aqueous stock solutions. Within 300 days no relevant degradation of linezolid or 2-ethoxybenzamide was detected. The stability of linezolid and 2-ethoxybenzamide after sample preparation is shown in figure 3. We did not detect degradation with any storage condition.

    Figure 2
    Figure 2

    Stability of linezolid and 2-ethoxybenzamide in aqueous stock solution at −32°C. The aqueous stock solutions are used as internal standard or quality control sample.

    Figure 3
    Figure 3

    Stability of linezolid and 2-ethoxybenzamide after sample preparation with acetonitrile/methanol at different storage conditions. CSF, cerebrospinal fluid.

    Quality control samples

    We performed QC samples to show our system and methods worked as we expected on days with analysis of unknown samples. Therefore, high and low concentration samples were prepared out of linezolid and 2-ethoxybenzamide stock solution with porcine serum. The low concentration sample was spiked with 2 mg/L linezolid and the high concentration sample was spiked with 10 mg/L linezolid. We defined the acceptable concentration range of linezolid with ±7% and the acceptable area range of 2-ethoxybenzamide with ±7.5% due to the recommendation of the European Medicines Agency guideline on bioanalytical method validation.7 They recommend ranges of ±15% but we decided to define closer limits with ±7% and ±7.5%. Of n=154 high concentration QC samples, only three missed the defined target range with mean linezolid concentration of 100 112% (96 698–106 944%) and mean 2-ethoxybenzamide area of 100 157% (87 996–106 621%). Of n=171 low concentration QC samples, 18 missed the defined target range with mean linezolid concentration of 100 072% (92 891–113 312%) and mean 2-ethoxybenzamide area of 100 090% (85 888–158 382%). The QC samples that missed the defined target ranges included handling mistakes during sample preparation.

    External validation

    To verify the performance of the methods an external validation assay was passed. This assay was offered by INSTAND (Gesellschaft zur Förderung der Qualitätssicherung in medizinischen Laboratorien e.V., Düsseldorf, Germany). The achieved certificate proves that two samples with unknown concentration of linezolid were analysed correctly within acceptable limits. The target concentration of sample 1 was 15.5 mg/L and we analysed 14.4 mg/L (deviation of −7.1%) within the acceptable limits from 10.9 mg/L to 20.2 mg/L. The target concentration of sample 2 was 22.4 mg/L and we analysed 21.0 mg/L (deviation of −6.3%) within the acceptable limits from 15.7 mg/L to 29.1 mg/L.

    Routine analysis

    The methods we described here have been routinely used in our laboratory to determine linezolid levels in human sera and CSF. Within the settings described above, we measured 20 pairs of simultaneous collected human serum and CSF samples from critically ill patients on intensive care units. The serum levels of linezolid ranged between 1.7 mg/L up to 34.9 mg/L (mean 8.2±7.1 mg/L, median 6.7±4.39 mg/L). The CSF levels of linezolid ranged between 0.9 mg/L up to 26.8 mg/L (mean 5.8±5.5 mg/L, median 6.7±4.39 mg/L). For our measurements the mean CSF/serum ratio was 0.71±0.16. Patient characteristics and dosage regimens are presented in table 2.

    View this table:
    Table 2

    Patient characteristics and dosage regimens

    Discussion

    We have demonstrated linezolid and 2-ethoxybenzamide to be stable in aqueous solution up to 1 year at −32°C. This is not surprising because it was shown that linezolid was stable when stored at 25°C for 34 days in commonly used intravenous fluids.10 Furthermore, linezolid and 2-ethoxybenzamide were stable after treatment with acetonitrile/methanol. This means that the prepared samples can be assayed within a 24 hour period without loss of linezolid or 2-ethoxybenzamide. The developed assays are reproducible, accurate, precise, and linear across the range of the calibration curves. The sample preparation of our methods is quick and simple. Performance of QC samples was solid with only 21 of 325 (6.5%) analyses missing the defined target range. Combined with the HPLC assay time of 15 min, this is ideal for the processing of samples for routine TDM. Previous studies described large interindividual variability in the concentrations of linezolid in plasma11 and CSF.12 Additionally variable results were published on whether linezolid plasma concentrations could be used as a surrogate parameter of CSF concentrations.12–15 Therefore, it is questionable whether the plasma and CSF concentrations of linezolid exceed the European Committee on Antimicrobial Susceptibility Testing (EUCAST) breakpoints for susceptible pathogens (4 mg/L).16 Especially in critically ill patients with central nervous system (CNS) infections caused by Gram-positive bacteria, the standard dosing regimen of linezolid 600 mg twice daily is considered unlikely to achieve optimal plasma and CSF concentrations.14 On the other hand, one case study of linezolid in meningitis reported similar drug concentrations in plasma and CSF, but this is unsurprising because drug penetration in inflamed meninges should be greater than in patients with non-inflamed meninges.13 These results push the need for TDM of linezolid in plasma and also potentially in CSF to avoid either the risk of dose-dependent toxicity or that of treatment failure.11

    The development of linezolid-induced thrombocytopenia is significantly affected in patients with a high serum linezolid concentration and renal impairment.17 Consistent evidence is now available showing that TDM and guided individual dose optimisation of linezolid is justified and feasible in clinical practice to improve tolerability and possibly response to therapy.18 Furthermore, different dosing strategies such as administration of higher than standard dosages or administration by continuous infusion should be considered.9 19 Another study showed that TDM may be useful to improve safety outcomes of adult patients with infections that require prolonged treatment with linezolid.20 The data from our routine analysis are in line with the other studies.

    The high variability of linezolid serum and CSF levels found in our study is remarkable. Although these patients received high doses of linezolid due to critical CNS infections, we measured insufficient linezolid levels in some of the CSF samples. In contrast to some other studies, we measured serum and CSF levels at the same time during continuous infusion of linezolid, providing us with an opportunity to calculate the CSF/serum ratio. For our 20 pairs of serum and CSF levels the mean ratio was 0.71±0.16. This means the CSF levels were 71±16% of the serum levels. Due to the small sample size, the 95% confidence interval for the mean blood–brain barrier penetration in our study was 0.64–0.78 (assuming normal distribution), but more data are required to estimate this more precisely. The main reason for the lack of samples was that we only collected CSF from patients with an external ventricular drain who received linezolid on our ICU.

    Conclusion

    In the present study, we developed simple methods for the quantification of linezolid in human serum and CSF. The developed methods could be easily and quickly performed and enabled the quantification of linezolid in patient samples for routine TDM. In the future, these methods could be used to evaluate the serum and CSF concentrations of linezolid in critically ill patients. Consequently, linezolid dosage regimens could be tailored to individual patients. Furthermore, the developed methods create the chance to study CSF penetration of linezolid because the simplest way to study the entry of drugs into the CNS is to measure drug concentrations in the CSF during a continuous drug infusion.21 Our investigation of the CSF penetration was limited due to the lack of more samples, but our results are in line with other studies that showed a high variability of serum and CSF levels. However, future studies can be performed using the methods described above.

    What this paper adds

    What is already known on this subject

    • Therapeutic drug monitoring (TDM) of antibiotics is highly recommended in intensive care units for critically ill patients.

    • Often TDM is focused on β-lactam antibiotics, but in times of increasing resistance other antibiotics of last resort such as linezolid deserve closer attention.

    What this study adds

    • This study adds HPLC-based assays for the quantification of linezolid that can be performed quickly and simply in hospital pharmacy settings.

    • The developed assays make it possible to study the penetration of linezolid into the central nervous system.

    • This study investigated 20 pairs of serum/cerebrospinal fluid levels collected at the same time during continuous infusion of linezolid.

    Data availability statement

    No data are available. Not applicable.

    Ethics statements

    Patient consent for publication

    Ethics approval

    This study involves human participants and was approved by “Ethikkommission der Ärztekammer Baden-Württemberg” in Stuttgart, Germany (authorisation number: F-2020-057). Participants gave informed consent to participate in the study before taking part.

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    Footnotes

    • EAHP Statement 4: Clinical Pharmacy Services.

    • Contributors SG and HV: development and validation of analytical assays. SG, SS, GG, HV and AR: collection of routine data. SG and HD: analysis and interpretation of data. SG, HV, AR, SS, LE, GG and HD: revision for intellectual content and approval of the final version. SG: acting as guarantor.

    • Funding The authors have not declared a specific grant for this research from any funding agency in the public, commercial or not-for-profit sectors.

    • Competing interests None declared.

    • Provenance and peer review Not commissioned; externally peer reviewed.