Overview of the analytical methods for vancomycin and/or teicoplanin determination in biological matrices
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paper_title: Simultaneous determination and stability studies of linezolid, meropenem and vancomycin in bacterial growth medium by high-performance liquid chromatography

paper_content:

Abstract For pharmacokinetic/pharmacodynamic (PK/PD) assessment of antibiotics combinations in in vitro infection models, accurate and precise quantification of drug concentrations in bacterial growth medium is crucial for derivation of valid PK/PD relationships. We aimed to (i) develop a high-performance liquid chromatography (HPLC) assay to simultaneously quantify linezolid (LZD), vancomycin (VAN) and meropenem (MER), as typical components of broad-spectrum antibiotic combination therapy, in bacterial growth medium cation-adjusted Mueller-Hinton broth (CaMHB) and (ii) determine the stability profiles of LZD, VAN and MER under conditions in in vitro infection models. To separate sample matrix components, the final method comprised the pretreatment of 100 μL sample with 400 μL methanol, the evaporation of supernatant and its reconstitution in water. A low sample volume of 2 μL processed sample was injected onto an Accucore C-18 column (2.6 μm, 100 × 2.1 mm) coupled to a Dionex Ultimate 3000 HPLC+ system. UV detection at 251, 240 and 302 nm allowed quantification limits of 0.5, 2 and 0.5 μg/mL for LZD, VAN and MER, respectively. The assay was successfully validated according to the relevant EMA guideline. The rapid method (14 min) was successfully applied to quantify significant degradation of LZD, VAN and MER in in vitro infection models: LZD was stable, VAN degraded to 90.6% and MER to 62.9% within 24 h compared to t = 0 in CaMHB at 37 °C, which should be considered when deriving PK/PD relationships in in vitro infection models. Inclusion of further antibiotics into the flexible gradient-based HPLC assay seems promising.
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paper_title: Methicillin and vancomycin resistant S. aureus in hospitalized patients

paper_content:

S. aureus is the major bacterial cause of skin, soft tissue and bone infections, and one of the commonest causes of healthcare-associated bacteremia. Hospital-associated methicillin-resistant S. aureus (MRSA) carriage is associated with an increased risk of infection, morbidity and mortality. Screening of high-risk patients at the time of hospital admission and decolonization has proved to be an important factor in an effort to reduce nosocomial transmission. The electronic database Pub Med was searched for all the articles on Establishment of MRSA and the emergence of vancomycin-resistant S. aureus (VRSA). The search included case reports, case series and reviews. All the articles were cross-referenced to search for any more available articles. A total of 88 references were obtained. The studies showed a steady increase in the number of vancomycin-intermediate and vancomycin-resistant S. aureus. Extensive use of vancomycin creates a selective pressure that favors the outgrowth of rare, vancomycin-resistant clones leading to heterogenous vancomycin intermediate S. aureus hVISA clones, and eventually, with continued exposure, to a uniform population of vancomycin-intermediate S. aureus (VISA) clones. However, the criteria for identifying hVISA strains have not been standardized, complicating any determination of their clinical significance and role in treatment failures. The spread of MRSA from the hospital to the community, coupled with the emergence of VISA and VRSA, has become major concern among healthcare providers. Infection-control measures, reliable laboratory screening for resistance, appropriate antibiotic prescribing practices and avoidance of blanket treatment can prevent long-term emergence of resistance.
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paper_title: Novel LC–MS/MS method for plasma vancomycin: Comparison with immunoassays and clinical impact

paper_content:

Abstract Background Accurate quantification of vancomycin in plasma is important for adequate dose-adjustment. As literature suggests between-method differences, our first objective was to develop a novel liquid chromatography–tandem mass spectrometry (LC–MS/MS) method for total vancomycin in human plasma and to compare frequently used immunoassays with this method. Secondly, we investigated the clinical impact of between-method quantification differences. Methods For LC–MS/MS, lithium heparin plasma was extracted by adding a precipitation reagent containing the internal standard (vancomycin-des-leucine). Analysis was performed on an Acquity TQD mass spectrometer equipped with an Acquity UPLC 2795 separations module. Our method was analytically validated and compared with four frequently used immunoassays from four different manufacturers. Vancomycin concentrations were clinically classified as toxic, therapeutic and sub-therapeutic. Clinical discordance was calculated using LC–MS/MS as a reference. Results A novel LC–MS/MS method using protein precipitation as sole pretreatment and an analysis time of 5.0 min was developed. The assay had a total imprecision of 2.6–8.5%, a limit of quantification of 0.3 mg/L and an accuracy ranging from 101.4 to 111.2%. Using LC–MS/MS as reference, three immunoassays showed a mean proportional difference within 10% and one showed a substantial mean proportional difference of > 20%. Clinical discordant interpretation of the obtained concentrations ranged from 6.1 to 22.2%. Conclusions We developed a novel LC–MS/MS method for rapid analysis of total vancomycin concentrations in human plasma. Correlation of the method with immunoassays showed a mean proportional difference > 20% for one of the assays, causing discordant clinical interpretation in more than 1 out of 5 samples.
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paper_title: Glycopeptide Antibiotics: Structure and Mechanisms of Action

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Glycopeptides of the clinically important antibiotic drugs are glycosylated cyclic or polycyclic nonribosomal peptides. Glycopeptides such as vancomycin and teicoplanin are often used for the treatment of gram-positive bacteria in patients. The increased incidence of drug resistance and inadequacy of these therapeutics against gram-positive bacterial infections would be the formation and clinical development of more variable second generation of glycopeptide antibiotics: semisynthetic lipoglycopeptide analogs such as telavancin, dalbavancin, and oritavancin with improved activity and better pharmacokinetic properties. In this review, we describe the development of and bacterial resistance to vancomycin, teicoplanin, and semisynthetic glycopeptides (teicoplanin, dalbavancin, and oritavancin). The clinical influence of resistance to glycopeptides, particularly vancomycin, are also discussed.
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paper_title: Simple and rapid quantification of vancomycin in serum, urine and peritoneal/pleural effusion via UHPLC–MS/MS applicable to personalized antibiotic dosing research

paper_content:

Abstract Management of the therapy of life-threatening bacterial infection is extremely based on an optimal antibiotic treatment. Achieving the correct vancomycin dosage in blood and target tissues can be complicated in special situations, e.g., where large fluid sequestration and/or acute renal failure occur. A UHPLC–MS/MS method operating in electrospray (ESI) positive ion mode was applied for the determination of vancomycin in serum, urine and peritoneal/pleural effusion. Sample pretreatment was composed of dilution and simple protein precipitation where only a small volume (50 μL) of serum, urine or peritoneal/pleural effusion was required. The separation of vancomycin was performed on a Meteoric Core C18 BIO column (100 × 4.6 mm, 2.7 μm) by gradient elution with 0.1% formic acid in water and acetonitrile. The total time of analysis was 4.5 min. The method was found to be linear in the range of 2–60 μM (or 0.5–10 μM) for serum, 0.27–10 μM (or 2–60 μM) for peritoneal/pleural effusion and 25–300 μM for urine, which was adequate for the determination of vancomycin in patient samples. The intra- and inter-day precision was below 8% RSD, and accuracy was from 89 to 104%. The UHPLC/MS–MS method offers a fast and reliable approach to determine vancomycin concentrations in three different human body fluid samples (serum, urine and peritoneal/pleural effusion) with a simple sample pretreatment that was the same for all selected specimens. This method should be applicable to large sample series in clinical (pharmacokinetic/pharmacodynamic) studies.
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paper_title: Comparative efficacy and safety of vancomycin versus teicoplanin: systematic review and meta-analysis.

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Vancomycin and teicoplanin are the glycopeptides currently in use for the treatment of infections caused by invasive beta-lactam-resistant gram-positive organisms. We conducted a systematic review and meta-analysis of randomized controlled trials that have compared vancomycin and teicoplanin administered systemically for the treatment of suspected or proven infections. A comprehensive search of trials without year, language, or publication status restrictions was performed. The primary outcome was all-cause mortality. Two reviewers independently extracted the data. Risk ratios (RRs) with 95% confidence intervals (CIs) were pooled by using the fixed-effect model (RRs of >1 favor vancomycin). Twenty-four trials were included. All-cause mortality was similar overall (RR, 0.95; 95% CI, 0.74 to 1.21), and there was no significant heterogeneity. In trials that used adequate allocation concealment, the results favored teicoplanin (RR, 0.82; 95% CI, 0.63 to 1.06), while in trials with unknown methods or inadequate concealment, the results favored vancomycin (RR, 3.61; 95% CI, 1.27 to 10.30). The latter trials might have recruited more severely ill patients. No other variable affected the RRs for mortality, including the assessment of glycopeptides administered empirically or for proven infections, neutropenia, the participant's age, and drug dosing. There were no significant differences between teicoplanin and vancomycin with regard to clinical failure (RR, 0.92; 95% CI, 0.81 to 1.05), microbiological failure (RR, 1.24; 95% CI, 0.93 to 1.65), and other efficacy outcomes. Lower RRs (in favor of teicoplanin) for clinical failure were observed with a lower risk of bias and when treatment was initiated for infections caused by gram-positive organisms rather than empirically. Total adverse events (RR, 0.61; 95% CI, 0.50 to 0.74), nephrotoxicity (RR, 0.44; 95% CI, 0.32 to 0.61), and red man syndrome were significantly less frequent with teicoplanin. Teicoplanin is not inferior to vancomycin with regard to efficacy and is associated with a lower adverse event rate than vancomycin.
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paper_title: Determination of the free and total concentrations of vancomycin by two-dimensional liquid chromatography and its application in elderly patients.

paper_content:

Abstract A robust two-dimensional liquid chromatography (2D-LC) method for determining the free and total concentrations of vancomycin in plasma was developed and validated. The 2D-LC system, which exhibited a strong capacity for inhibiting interference, comprised a unique RP1–IEX–RP2 column system and an “Assistant Flow” configuration. Ultrafiltration technology was employed to separate free vancomycin from the protein-bound fraction in human plasma. The influence of ultrafiltration conditions on the free vancomycin concentration was evaluated. The calibration curve was linear over the 0.195–49.92 μg/ml range for the free and total vancomycin concentrations. The within- and between-run precision ranges were 1.5–3.9% and 2.0–4.7% for the total concentration, 1.4–3.3% and 2.4–4.0% for the free concentration, respectively. Ultrafiltration was susceptible to variations in the experimental conditions, including the centrifugation time, the centrifugal force, and the nominal molecular weight limit of the ultrafiltration membrane. A total of 101 serum samples from 84 elderly patients were analyzed by this method. The free vancomycin concentration was 5.88 ± 3.75 μg/ml (range: 0.240–16.79 μg/ml), the total concentration was 12.36 ± 5.36 μg/ml (range: 2.16–27.14 μg/ml), and the unbound fraction was 45.6 ± 18.8% (range: 11.1–96.9%). There was a poor correlation between the free and total vancomycin concentrations (R2 = 0.596, p
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paper_title: New liquid chromatography-tandem mass spectrometry method for routine TDM of vancomycin in patients with both normal and impaired renal functions and comparison with results of polarization fluoroimmunoassay in light of varying creatinine concentrations

paper_content:

Abstract A new LC-MS/MS method with simple sample extraction and a relatively short period of vancomycin analysis for routine therapeutic drug monitoring was developed and validated. 50 μL serum was precipitated using 20 μL 33% trichloroacetic acid and 0.5 mol/L NH4OH was added to increase pH before analysis. A RP BEH C18, 1.7 μm, 2.1 × 50 mm column maintained at 30 °C and tobramycin as internal standard were used. Mass detection was performed in positive electrospray mode. The results obtained with LC-MS/MS method were correlated with an FPIA assay (Abbott AxSYM) using mouse monoclonal antibody. Subjects were divided into three groups according to creatinine levels (53.5 ± 19.1, 150.2 ± 48.4, 471.7 ± 124.7 μmol/L) and Passing-Bablok regression analysis and Bland-Altman analysis were used to compare vancomycin concentrations. The results of subjects with both normal and higher creatinine levels correlated very well and the linear regression model equations were near ideal (LC-MSVAN = 0.947 × AbbottVAN + 0.192 and LC-MSVAN = 0.973 × AbbottVAN − 0.411 respectively). Dialyzed patients with the highest creatinine levels showed about 14% greater vancomycin concentration with the FPIA assay (LC-MSVAN = 0.866 × AbbottVAN + 2.127). This overestimation probably due to the presence of the metabolite CDP ought not to be of clinical relevance owing to the wide range of recommended vancomycin concentration.
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paper_title: Development and validation of a new ultra-performance liquid chromatographic method for vancomycin assay in serum and its application to therapeutic drug monitoring.

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OBJECTIVE ::: The aim of this study was to develop and validate an ultra-performance liquid chromatographic (UPLC) method with photodiode array detector for the measurement of vancomycin in human serum samples for therapeutic drug monitoring or other applications. ::: ::: ::: METHODS ::: The method included the extraction of vancomycin in serum by deproteinization with acetonitrile. The analyses were carried out using an ACQUITY UPLC BEH C(18) column (2.1 × 50 mm, 1.7 μm) using acetonitrile and 0.005 M KH(2)PO(4) buffer (pH 2.5) as the mobile phase at a flow rate of 0.3 mL/min, with photodiode array detection at 230 nm. The method was validated for extraction recovery, inter- and intraday precision (relative standard deviation, RSD%), and accuracy and stability of vancomycin in serum. Both the established UPLC method and fluorescence polarization immunoassay (FPIA) were used to measure the prepared quality control (QC) samples (1.0, 7.0, 35.0, 75.0 mg/L) to validate the accuracy of UPLC. Furthermore, both methods were subsequently used to assay the vancomycin concentration in 172 clinical serum samples collected from patients receiving vancomycin in the hospitals localized in Shanghai (China) and 32 control samples from United Kingdom National External Quality Assessment Service (UK NEQAS). ::: ::: ::: RESULTS ::: The retention time of vancomycin was 2.6 minutes. The calibration curve for UPLC was linear over the range 1.0-100.0 mg/L (R(2) > 0.999). The method was fully validated in terms of recovery, selectivity, accuracy, precision, and various conditions. The absolute difference% and RSD% of the prepared QC samples assayed by UPLC were all better than the results by FPIA. A paired t test of the results of the prepared QC samples indicated that the results of all the QC samples had significant difference (P < 0.05), except for the 7.0 mg/L QC samples, which suggested that UPLC was more accurate for the samples containing low or high concentration of vancomycin. A correlation with the Deming model provided a good linear relation between the results of the 2 methods applied to 172 samples, with equation of UPLC = 0.99 × FPIA - 0.19 (R(2)= 0.923), and the agreement of the 2 methods was illustrated using Bland-Altman plot with a mean difference (UPLC - FPIA) of -0.428 mg/L and 95% confidence interval of -8.33 to 7.47 mg/L, respectively. A Student t test comparing results obtained by the UPLC method and group mean results of control samples from UK NEQAS were not significant (P = 0.057). ::: ::: ::: CONCLUSIONS ::: A short analysis time, small amount of serum needed, high specificity, and accuracy make the UPLC method developed in this study appropriate and practical for vancomycin therapeutic drug monitoring and could be applied to other nonserum applications or where requiring superior validation parameters such as for pharmacokinetic/pharmacodynamic studies.
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paper_title: An ultra-high pressure liquid chromatography-tandem mass spectrometry method for the quantification of teicoplanin in plasma of neonates.

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The development and validation of an ultra-high pressure liquid chromatography (UHPLC) tandem mass spectrometry (MS/MS) method was performed with the aim to be applied for the quantification of plasma teicoplanin concentrations in neonates. Pharmacokinetic data of teicoplanin in the neonatal population is very limited, therefore, a sensitive and reliable method for the determination of all isoforms of teicoplanin applied in a low volume of sample is of real importance. Teicoplanin main components were extracted by a simple acetonitrile precipitation step and analysed on a C18 chromatographic column by a triple quadrupole MS with electrospray ionization. The method provides quantitative data over a linear range of 25-6400ng/mL with LOD 8.5ng/mL and LOQ 25ng/mL for total teicoplanin. The method was applied in plasma samples from neonates to support pharmacokinetic data and proved to be a reliable and fast method for the quantification of teicoplanin concentration levels in plasma of infants during therapy in Intensive Care Unit.
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paper_title: Simultaneous analysis of antibiotics in biological samples by ultra high performance liquid chromatography–tandem mass spectrometry

paper_content:

Abstract A rapid and reliable multiclass method was developed for the simultaneous analysis of 21 antibiotics (beta-lactams, aminoglycosides, penicillins, cephalosporins, carbapenems or quinolones) in urine, serum, cerebrospinal fluid (CSF) and bronchial aspirations by ultra-high performance liquid chromatography coupled to tandem mass spectrometry (UHPLC–MS/MS). Prior to chromatographic determination, the analytes were extracted from human biological fluids by simple sample treatments, which imply dilution, liquefaction, or protein precipitation. Several chromatographic conditions were optimized in order to obtain a fast separation (
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paper_title: Modern methods for vancomycin determination in biological fluids by methods based on high-performance liquid chromatography--A review.

paper_content:

Vancomycin is a glycopeptide antibiotic used in the therapy of severe bacterial infection. The monitoring of vancomycin levels is recommended because of its narrow therapeutic index and toxicity. This measurement is especially appropriate in patients with unstable renal functions, who receive high doses of vancomycin or present serious bacterial infections accompanied by important sequestration of liquids when it could be difficult to achieve the optimal therapeutic dose. Most of the methods for vancomycin determination in routine practice are immunoassays. However, chromatography-based techniques in combination with UV or mass spectrometry detection provide results with greater accuracy and precision also in complicated biological matrices. This review provides a detailed overview of modern approaches for the chromatographic separation of vancomycin in various biological samples and useful sample preparation procedures for vancomycin determination in various biological fluids.
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paper_title: High-throughput determination of vancomycin in human plasma by a cost-effective system of two-dimensional liquid chromatography.

paper_content:

Therapeutic drug monitoring (TDM) is one of the most important services of clinical laboratories. Two main techniques are commonly used: the immunoassay and chromatography method. We have developed a cost-effective system of two-dimensional liquid chromatography with ultraviolet detection (2D-LC-UV) for high-throughput determination of vancomycin in human plasma that combines the automation and low start-up costs of the immunoassay with the high selectivity and sensitivity of the liquid chromatography coupled with mass spectrometric detection without incurring their disadvantages, achieving high cost-effectiveness. This 2D-LC system offers a large volume injection to provide sufficient sensitivity and uses simulated gradient peak compression technology to control peak broadening and to improve peak shape. A middle column was added to reduce the analysis cycle time and make it suitable for high-throughput routine clinical assays. The analysis cycle time was 4min and the peak width was 0.8min. Compared with other chromatographic methods that have been developed, the analysis cycle time and peak width for vancomycin was reduced significantly. The lower limit of quantification was 0.20μg/mL for vancomycin, which is the same as certain LC-MS/MS methods that have been recently developed and validated. The method is rapid, automated, and low-cost and has high selectivity and sensitivity for the quantification of vancomycin in human plasma, thus making it well-suited for use in hospital clinical laboratories.
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paper_title: A simple quantitative method analysing amikacin, gentamicin, and vancomycin levels in human newborn plasma using ion-pair liquid chromatography/tandem mass spectrometry and its applicability to a clinical study.

paper_content:

Neuroprotective controlled therapeutic hypothermia is the standard of care for newborns suffering perinatal asphyxia. Antibiotic drugs, such as amikacin, gentamicin, and vancomycin are frequently administered during controlled hypothermia, which possibly alters their pharmacokinetic (PK) and pharmacodynamic (PD) profiles. In order to examine this effect an LC-MS/MS method for the simultaneous quantification of amikacin, the major gentamicin components (gentamicin C, C1a and C2), and vancomycin in plasma was developed. In 25μL plasma proteins were precipitated with trichloroacetic acid (TCA) and detection of the components was achieved using ion-pair reversed phase chromatography coupled with electrospray ionization tandem mass spectrometry. The chromatographic runtime was 7.5min per sample. Calibration standards were prepared over a range of 0.3-50mgL(-1) for amikacin and gentamicin and 1.0-100mgL(-1) for vancomycin. At LLOQ accuracy was between 103 and 120% and imprecision was less than 19%. For concentrations above LLOQ accuracy ranged from 98% to 102% and imprecision was less than 6%. Process efficiency, ionization efficiency, and recovery were acceptable. Samples and stock solutions were stable during the time periods and at the different temperatures examined. The applicability of the method was shown by analysing plasma samples from 3 neonatal patients. The developed method allows accurate and precise simultaneous quantification of amikacin, gentamicin, and vancomycin in a small volume (25μL) of plasma.
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paper_title: Accuracy of the analysis of free vancomycin concentration by ultrafiltration in various disease states

paper_content:

In the present study, the accuracy of the analysis of free vancomycin (VCM) by ultrafiltration in various disease conditions was assessed. With VCM as a representative drug, we used clinical plasma samples to investigate the effect of plasma conditions on the volume ratio of ultrafiltrate to sample solution (Vu/Vs) and the consequential effect on the measured free drug concentration (fc). Our results demonstrated that plasma conditions had a significant impact on Vu/Vs by centrifugal ultrafiltration (CF-UF). The Vu by CF-UF ranged from 97 μL to 279 μL among different individuals under the same centrifugation conditions. Total protein levels and the osmotic pressure of plasma were the main influence factors of Vu/Vs in disease states. In contrast, the Vu/Vs by hollow fiber centrifugal ultrafiltration (HFCF-UF) was less influenced by plasma conditions. As a consequence, the results of fc determined by HFCF-UF were more accurate than those by CF-UF for patients with different disease states. HFCF-UF displayed great advantages in clinical samples for accurate analysis of fc. It has been successfully applied to monitor free VCM in clinical plasma samples in routine therapeutic drug monitoring (TDM).
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paper_title: Determination of Vancomycin in Human Plasma, Bone and Fat by Liquid Chromatography/Tandem Mass Spectrometry

paper_content:

A liquid chromatography/tandem mass spectrometry (LC-MS/MS) assay was developed for the determination of vancomycin in human plasma, bone and fat tissue. For vancomycin in plasma, sample was treated with methanol to precipitate the proteins. After centrifugation, the supernatant was diluted with water, and then injected into the LC-MS/MS system. For vancomycin in bone and fat, the pulverized bone/fat samples were immersed in phosphate buffered saline pH 7.3 at 4°C overnight. After centrifugation, the supernatant of bone/fat tissue suspension was treated in the same way as the plasma samples. Vancomycin and aminopterin, the internal standard, were resolved on a C18(2) column using gradient elution of 0.05% formic acid and methanol. The two compounds were detected using electrospray ionisation in the positive mode. Standard curves were linear over the concentration range 0.05 to 50 mg/L (r2>0.99) in plasma, bone and fat. Bias was ≤ ± 10% from 0.05 to 50 mg/L, intra- and inter-day coefficients of variation (imprecision) were <10%, and the limit of quantification was 0.05 mg/L. The assay has been used successfully in a clinical study to investigate the regional delivery of vancomycin in bone and fat after prophylactic administration of vancomycin through an intraosseous route or systemic route, during total knee arthroplasty.
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paper_title: Direct injection LC-MS/MS method for the determination of teicoplanin in human plasma.

paper_content:

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 0.5mL/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 1-50mg/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.
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paper_title: Simultaneous determination and stability studies of linezolid, meropenem and vancomycin in bacterial growth medium by high-performance liquid chromatography

paper_content:

Abstract For pharmacokinetic/pharmacodynamic (PK/PD) assessment of antibiotics combinations in in vitro infection models, accurate and precise quantification of drug concentrations in bacterial growth medium is crucial for derivation of valid PK/PD relationships. We aimed to (i) develop a high-performance liquid chromatography (HPLC) assay to simultaneously quantify linezolid (LZD), vancomycin (VAN) and meropenem (MER), as typical components of broad-spectrum antibiotic combination therapy, in bacterial growth medium cation-adjusted Mueller-Hinton broth (CaMHB) and (ii) determine the stability profiles of LZD, VAN and MER under conditions in in vitro infection models. To separate sample matrix components, the final method comprised the pretreatment of 100 μL sample with 400 μL methanol, the evaporation of supernatant and its reconstitution in water. A low sample volume of 2 μL processed sample was injected onto an Accucore C-18 column (2.6 μm, 100 × 2.1 mm) coupled to a Dionex Ultimate 3000 HPLC+ system. UV detection at 251, 240 and 302 nm allowed quantification limits of 0.5, 2 and 0.5 μg/mL for LZD, VAN and MER, respectively. The assay was successfully validated according to the relevant EMA guideline. The rapid method (14 min) was successfully applied to quantify significant degradation of LZD, VAN and MER in in vitro infection models: LZD was stable, VAN degraded to 90.6% and MER to 62.9% within 24 h compared to t = 0 in CaMHB at 37 °C, which should be considered when deriving PK/PD relationships in in vitro infection models. Inclusion of further antibiotics into the flexible gradient-based HPLC assay seems promising.
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paper_title: Novel LC–MS/MS method for plasma vancomycin: Comparison with immunoassays and clinical impact

paper_content:

Abstract Background Accurate quantification of vancomycin in plasma is important for adequate dose-adjustment. As literature suggests between-method differences, our first objective was to develop a novel liquid chromatography–tandem mass spectrometry (LC–MS/MS) method for total vancomycin in human plasma and to compare frequently used immunoassays with this method. Secondly, we investigated the clinical impact of between-method quantification differences. Methods For LC–MS/MS, lithium heparin plasma was extracted by adding a precipitation reagent containing the internal standard (vancomycin-des-leucine). Analysis was performed on an Acquity TQD mass spectrometer equipped with an Acquity UPLC 2795 separations module. Our method was analytically validated and compared with four frequently used immunoassays from four different manufacturers. Vancomycin concentrations were clinically classified as toxic, therapeutic and sub-therapeutic. Clinical discordance was calculated using LC–MS/MS as a reference. Results A novel LC–MS/MS method using protein precipitation as sole pretreatment and an analysis time of 5.0 min was developed. The assay had a total imprecision of 2.6–8.5%, a limit of quantification of 0.3 mg/L and an accuracy ranging from 101.4 to 111.2%. Using LC–MS/MS as reference, three immunoassays showed a mean proportional difference within 10% and one showed a substantial mean proportional difference of > 20%. Clinical discordant interpretation of the obtained concentrations ranged from 6.1 to 22.2%. Conclusions We developed a novel LC–MS/MS method for rapid analysis of total vancomycin concentrations in human plasma. Correlation of the method with immunoassays showed a mean proportional difference > 20% for one of the assays, causing discordant clinical interpretation in more than 1 out of 5 samples.
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paper_title: Simple and rapid quantification of vancomycin in serum, urine and peritoneal/pleural effusion via UHPLC–MS/MS applicable to personalized antibiotic dosing research

paper_content:

Abstract Management of the therapy of life-threatening bacterial infection is extremely based on an optimal antibiotic treatment. Achieving the correct vancomycin dosage in blood and target tissues can be complicated in special situations, e.g., where large fluid sequestration and/or acute renal failure occur. A UHPLC–MS/MS method operating in electrospray (ESI) positive ion mode was applied for the determination of vancomycin in serum, urine and peritoneal/pleural effusion. Sample pretreatment was composed of dilution and simple protein precipitation where only a small volume (50 μL) of serum, urine or peritoneal/pleural effusion was required. The separation of vancomycin was performed on a Meteoric Core C18 BIO column (100 × 4.6 mm, 2.7 μm) by gradient elution with 0.1% formic acid in water and acetonitrile. The total time of analysis was 4.5 min. The method was found to be linear in the range of 2–60 μM (or 0.5–10 μM) for serum, 0.27–10 μM (or 2–60 μM) for peritoneal/pleural effusion and 25–300 μM for urine, which was adequate for the determination of vancomycin in patient samples. The intra- and inter-day precision was below 8% RSD, and accuracy was from 89 to 104%. The UHPLC/MS–MS method offers a fast and reliable approach to determine vancomycin concentrations in three different human body fluid samples (serum, urine and peritoneal/pleural effusion) with a simple sample pretreatment that was the same for all selected specimens. This method should be applicable to large sample series in clinical (pharmacokinetic/pharmacodynamic) studies.
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paper_title: New liquid chromatography-tandem mass spectrometry method for routine TDM of vancomycin in patients with both normal and impaired renal functions and comparison with results of polarization fluoroimmunoassay in light of varying creatinine concentrations

paper_content:

Abstract A new LC-MS/MS method with simple sample extraction and a relatively short period of vancomycin analysis for routine therapeutic drug monitoring was developed and validated. 50 μL serum was precipitated using 20 μL 33% trichloroacetic acid and 0.5 mol/L NH4OH was added to increase pH before analysis. A RP BEH C18, 1.7 μm, 2.1 × 50 mm column maintained at 30 °C and tobramycin as internal standard were used. Mass detection was performed in positive electrospray mode. The results obtained with LC-MS/MS method were correlated with an FPIA assay (Abbott AxSYM) using mouse monoclonal antibody. Subjects were divided into three groups according to creatinine levels (53.5 ± 19.1, 150.2 ± 48.4, 471.7 ± 124.7 μmol/L) and Passing-Bablok regression analysis and Bland-Altman analysis were used to compare vancomycin concentrations. The results of subjects with both normal and higher creatinine levels correlated very well and the linear regression model equations were near ideal (LC-MSVAN = 0.947 × AbbottVAN + 0.192 and LC-MSVAN = 0.973 × AbbottVAN − 0.411 respectively). Dialyzed patients with the highest creatinine levels showed about 14% greater vancomycin concentration with the FPIA assay (LC-MSVAN = 0.866 × AbbottVAN + 2.127). This overestimation probably due to the presence of the metabolite CDP ought not to be of clinical relevance owing to the wide range of recommended vancomycin concentration.
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paper_title: Development and validation of a new ultra-performance liquid chromatographic method for vancomycin assay in serum and its application to therapeutic drug monitoring.

paper_content:

OBJECTIVE ::: The aim of this study was to develop and validate an ultra-performance liquid chromatographic (UPLC) method with photodiode array detector for the measurement of vancomycin in human serum samples for therapeutic drug monitoring or other applications. ::: ::: ::: METHODS ::: The method included the extraction of vancomycin in serum by deproteinization with acetonitrile. The analyses were carried out using an ACQUITY UPLC BEH C(18) column (2.1 × 50 mm, 1.7 μm) using acetonitrile and 0.005 M KH(2)PO(4) buffer (pH 2.5) as the mobile phase at a flow rate of 0.3 mL/min, with photodiode array detection at 230 nm. The method was validated for extraction recovery, inter- and intraday precision (relative standard deviation, RSD%), and accuracy and stability of vancomycin in serum. Both the established UPLC method and fluorescence polarization immunoassay (FPIA) were used to measure the prepared quality control (QC) samples (1.0, 7.0, 35.0, 75.0 mg/L) to validate the accuracy of UPLC. Furthermore, both methods were subsequently used to assay the vancomycin concentration in 172 clinical serum samples collected from patients receiving vancomycin in the hospitals localized in Shanghai (China) and 32 control samples from United Kingdom National External Quality Assessment Service (UK NEQAS). ::: ::: ::: RESULTS ::: The retention time of vancomycin was 2.6 minutes. The calibration curve for UPLC was linear over the range 1.0-100.0 mg/L (R(2) > 0.999). The method was fully validated in terms of recovery, selectivity, accuracy, precision, and various conditions. The absolute difference% and RSD% of the prepared QC samples assayed by UPLC were all better than the results by FPIA. A paired t test of the results of the prepared QC samples indicated that the results of all the QC samples had significant difference (P < 0.05), except for the 7.0 mg/L QC samples, which suggested that UPLC was more accurate for the samples containing low or high concentration of vancomycin. A correlation with the Deming model provided a good linear relation between the results of the 2 methods applied to 172 samples, with equation of UPLC = 0.99 × FPIA - 0.19 (R(2)= 0.923), and the agreement of the 2 methods was illustrated using Bland-Altman plot with a mean difference (UPLC - FPIA) of -0.428 mg/L and 95% confidence interval of -8.33 to 7.47 mg/L, respectively. A Student t test comparing results obtained by the UPLC method and group mean results of control samples from UK NEQAS were not significant (P = 0.057). ::: ::: ::: CONCLUSIONS ::: A short analysis time, small amount of serum needed, high specificity, and accuracy make the UPLC method developed in this study appropriate and practical for vancomycin therapeutic drug monitoring and could be applied to other nonserum applications or where requiring superior validation parameters such as for pharmacokinetic/pharmacodynamic studies.
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paper_title: An ultra-high pressure liquid chromatography-tandem mass spectrometry method for the quantification of teicoplanin in plasma of neonates.

paper_content:

The development and validation of an ultra-high pressure liquid chromatography (UHPLC) tandem mass spectrometry (MS/MS) method was performed with the aim to be applied for the quantification of plasma teicoplanin concentrations in neonates. Pharmacokinetic data of teicoplanin in the neonatal population is very limited, therefore, a sensitive and reliable method for the determination of all isoforms of teicoplanin applied in a low volume of sample is of real importance. Teicoplanin main components were extracted by a simple acetonitrile precipitation step and analysed on a C18 chromatographic column by a triple quadrupole MS with electrospray ionization. The method provides quantitative data over a linear range of 25-6400ng/mL with LOD 8.5ng/mL and LOQ 25ng/mL for total teicoplanin. The method was applied in plasma samples from neonates to support pharmacokinetic data and proved to be a reliable and fast method for the quantification of teicoplanin concentration levels in plasma of infants during therapy in Intensive Care Unit.
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paper_title: Simultaneous analysis of antibiotics in biological samples by ultra high performance liquid chromatography–tandem mass spectrometry

paper_content:

Abstract A rapid and reliable multiclass method was developed for the simultaneous analysis of 21 antibiotics (beta-lactams, aminoglycosides, penicillins, cephalosporins, carbapenems or quinolones) in urine, serum, cerebrospinal fluid (CSF) and bronchial aspirations by ultra-high performance liquid chromatography coupled to tandem mass spectrometry (UHPLC–MS/MS). Prior to chromatographic determination, the analytes were extracted from human biological fluids by simple sample treatments, which imply dilution, liquefaction, or protein precipitation. Several chromatographic conditions were optimized in order to obtain a fast separation (
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paper_title: High-throughput determination of vancomycin in human plasma by a cost-effective system of two-dimensional liquid chromatography.

paper_content:

Therapeutic drug monitoring (TDM) is one of the most important services of clinical laboratories. Two main techniques are commonly used: the immunoassay and chromatography method. We have developed a cost-effective system of two-dimensional liquid chromatography with ultraviolet detection (2D-LC-UV) for high-throughput determination of vancomycin in human plasma that combines the automation and low start-up costs of the immunoassay with the high selectivity and sensitivity of the liquid chromatography coupled with mass spectrometric detection without incurring their disadvantages, achieving high cost-effectiveness. This 2D-LC system offers a large volume injection to provide sufficient sensitivity and uses simulated gradient peak compression technology to control peak broadening and to improve peak shape. A middle column was added to reduce the analysis cycle time and make it suitable for high-throughput routine clinical assays. The analysis cycle time was 4min and the peak width was 0.8min. Compared with other chromatographic methods that have been developed, the analysis cycle time and peak width for vancomycin was reduced significantly. The lower limit of quantification was 0.20μg/mL for vancomycin, which is the same as certain LC-MS/MS methods that have been recently developed and validated. The method is rapid, automated, and low-cost and has high selectivity and sensitivity for the quantification of vancomycin in human plasma, thus making it well-suited for use in hospital clinical laboratories.
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paper_title: A simple quantitative method analysing amikacin, gentamicin, and vancomycin levels in human newborn plasma using ion-pair liquid chromatography/tandem mass spectrometry and its applicability to a clinical study.

paper_content:

Neuroprotective controlled therapeutic hypothermia is the standard of care for newborns suffering perinatal asphyxia. Antibiotic drugs, such as amikacin, gentamicin, and vancomycin are frequently administered during controlled hypothermia, which possibly alters their pharmacokinetic (PK) and pharmacodynamic (PD) profiles. In order to examine this effect an LC-MS/MS method for the simultaneous quantification of amikacin, the major gentamicin components (gentamicin C, C1a and C2), and vancomycin in plasma was developed. In 25μL plasma proteins were precipitated with trichloroacetic acid (TCA) and detection of the components was achieved using ion-pair reversed phase chromatography coupled with electrospray ionization tandem mass spectrometry. The chromatographic runtime was 7.5min per sample. Calibration standards were prepared over a range of 0.3-50mgL(-1) for amikacin and gentamicin and 1.0-100mgL(-1) for vancomycin. At LLOQ accuracy was between 103 and 120% and imprecision was less than 19%. For concentrations above LLOQ accuracy ranged from 98% to 102% and imprecision was less than 6%. Process efficiency, ionization efficiency, and recovery were acceptable. Samples and stock solutions were stable during the time periods and at the different temperatures examined. The applicability of the method was shown by analysing plasma samples from 3 neonatal patients. The developed method allows accurate and precise simultaneous quantification of amikacin, gentamicin, and vancomycin in a small volume (25μL) of plasma.
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paper_title: Determination of Vancomycin in Human Plasma, Bone and Fat by Liquid Chromatography/Tandem Mass Spectrometry

paper_content:

A liquid chromatography/tandem mass spectrometry (LC-MS/MS) assay was developed for the determination of vancomycin in human plasma, bone and fat tissue. For vancomycin in plasma, sample was treated with methanol to precipitate the proteins. After centrifugation, the supernatant was diluted with water, and then injected into the LC-MS/MS system. For vancomycin in bone and fat, the pulverized bone/fat samples were immersed in phosphate buffered saline pH 7.3 at 4°C overnight. After centrifugation, the supernatant of bone/fat tissue suspension was treated in the same way as the plasma samples. Vancomycin and aminopterin, the internal standard, were resolved on a C18(2) column using gradient elution of 0.05% formic acid and methanol. The two compounds were detected using electrospray ionisation in the positive mode. Standard curves were linear over the concentration range 0.05 to 50 mg/L (r2>0.99) in plasma, bone and fat. Bias was ≤ ± 10% from 0.05 to 50 mg/L, intra- and inter-day coefficients of variation (imprecision) were <10%, and the limit of quantification was 0.05 mg/L. The assay has been used successfully in a clinical study to investigate the regional delivery of vancomycin in bone and fat after prophylactic administration of vancomycin through an intraosseous route or systemic route, during total knee arthroplasty.
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paper_title: Accuracy of the analysis of free vancomycin concentration by ultrafiltration in various disease states

paper_content:

In the present study, the accuracy of the analysis of free vancomycin (VCM) by ultrafiltration in various disease conditions was assessed. With VCM as a representative drug, we used clinical plasma samples to investigate the effect of plasma conditions on the volume ratio of ultrafiltrate to sample solution (Vu/Vs) and the consequential effect on the measured free drug concentration (fc). Our results demonstrated that plasma conditions had a significant impact on Vu/Vs by centrifugal ultrafiltration (CF-UF). The Vu by CF-UF ranged from 97 μL to 279 μL among different individuals under the same centrifugation conditions. Total protein levels and the osmotic pressure of plasma were the main influence factors of Vu/Vs in disease states. In contrast, the Vu/Vs by hollow fiber centrifugal ultrafiltration (HFCF-UF) was less influenced by plasma conditions. As a consequence, the results of fc determined by HFCF-UF were more accurate than those by CF-UF for patients with different disease states. HFCF-UF displayed great advantages in clinical samples for accurate analysis of fc. It has been successfully applied to monitor free VCM in clinical plasma samples in routine therapeutic drug monitoring (TDM).
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paper_title: Direct injection LC-MS/MS method for the determination of teicoplanin in human plasma.

paper_content:

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 0.5mL/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 1-50mg/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.
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paper_title: Simultaneous determination and stability studies of linezolid, meropenem and vancomycin in bacterial growth medium by high-performance liquid chromatography

paper_content:

Abstract For pharmacokinetic/pharmacodynamic (PK/PD) assessment of antibiotics combinations in in vitro infection models, accurate and precise quantification of drug concentrations in bacterial growth medium is crucial for derivation of valid PK/PD relationships. We aimed to (i) develop a high-performance liquid chromatography (HPLC) assay to simultaneously quantify linezolid (LZD), vancomycin (VAN) and meropenem (MER), as typical components of broad-spectrum antibiotic combination therapy, in bacterial growth medium cation-adjusted Mueller-Hinton broth (CaMHB) and (ii) determine the stability profiles of LZD, VAN and MER under conditions in in vitro infection models. To separate sample matrix components, the final method comprised the pretreatment of 100 μL sample with 400 μL methanol, the evaporation of supernatant and its reconstitution in water. A low sample volume of 2 μL processed sample was injected onto an Accucore C-18 column (2.6 μm, 100 × 2.1 mm) coupled to a Dionex Ultimate 3000 HPLC+ system. UV detection at 251, 240 and 302 nm allowed quantification limits of 0.5, 2 and 0.5 μg/mL for LZD, VAN and MER, respectively. The assay was successfully validated according to the relevant EMA guideline. The rapid method (14 min) was successfully applied to quantify significant degradation of LZD, VAN and MER in in vitro infection models: LZD was stable, VAN degraded to 90.6% and MER to 62.9% within 24 h compared to t = 0 in CaMHB at 37 °C, which should be considered when deriving PK/PD relationships in in vitro infection models. Inclusion of further antibiotics into the flexible gradient-based HPLC assay seems promising.
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paper_title: Novel LC–MS/MS method for plasma vancomycin: Comparison with immunoassays and clinical impact

paper_content:

Abstract Background Accurate quantification of vancomycin in plasma is important for adequate dose-adjustment. As literature suggests between-method differences, our first objective was to develop a novel liquid chromatography–tandem mass spectrometry (LC–MS/MS) method for total vancomycin in human plasma and to compare frequently used immunoassays with this method. Secondly, we investigated the clinical impact of between-method quantification differences. Methods For LC–MS/MS, lithium heparin plasma was extracted by adding a precipitation reagent containing the internal standard (vancomycin-des-leucine). Analysis was performed on an Acquity TQD mass spectrometer equipped with an Acquity UPLC 2795 separations module. Our method was analytically validated and compared with four frequently used immunoassays from four different manufacturers. Vancomycin concentrations were clinically classified as toxic, therapeutic and sub-therapeutic. Clinical discordance was calculated using LC–MS/MS as a reference. Results A novel LC–MS/MS method using protein precipitation as sole pretreatment and an analysis time of 5.0 min was developed. The assay had a total imprecision of 2.6–8.5%, a limit of quantification of 0.3 mg/L and an accuracy ranging from 101.4 to 111.2%. Using LC–MS/MS as reference, three immunoassays showed a mean proportional difference within 10% and one showed a substantial mean proportional difference of > 20%. Clinical discordant interpretation of the obtained concentrations ranged from 6.1 to 22.2%. Conclusions We developed a novel LC–MS/MS method for rapid analysis of total vancomycin concentrations in human plasma. Correlation of the method with immunoassays showed a mean proportional difference > 20% for one of the assays, causing discordant clinical interpretation in more than 1 out of 5 samples.
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paper_title: Simultaneous quantification of antimicrobial agents for multidrug-resistant bacterial infections in human plasma by ultra-high-pressure liquid chromatography-tandem mass spectrometry.

paper_content:

Antibiotic-resistant bacterial infection is one of the most serious clinical problems worldwide. Vancomycin, teicoplanin, daptomycin, and colistin are glycopeptide and lipopeptide antibiotics that are frequently used to treat multidrug-resistant bacterial infections. Therapeutic drug monitoring is recommended to ensure both safety and efficacy and to improve clinical outcomes. This study developed a fast, simple, and sensitive ultra-high-pressure liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method for the simultaneous determination of the concentrations of these four drugs in human plasma. The sample preparation process includes a simple protein denaturation step using acetonitrile, followed by an 11-fold dilution with 0.1% formic acid. Eight target peaks for the four drugs can be analyzed within 3 min using a Kinetex™ 2.6 μm C18 column. The mass spectrometry parameters were optimized, and two transitions for each target peak were used for multiple reaction monitoring, which provided high sensitivity and specificity. The UHPLC-MS/MS method was validated over clinical concentration ranges. The intra-day and inter-day precisions for the ratio of the peak area of each analyte to the peak area of the internal standard were all below 12.7 and 14.7% relative standard deviations, respectively. The accuracy at low, medium, and high concentrations of the eight target peaks was between 89.3 and 110.7%. The standard curves for the analytes were linear and had coefficients of determination higher than 0.997. The limits of detection were all below 70 ng mL(-1). The use of this method to analyze patient plasma samples confirmed that it is effective for the therapeutic drug monitoring of these four drugs and can be used to improve the therapeutic efficacy and safety of treatment with antibiotics.
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paper_title: Simple and rapid quantification of vancomycin in serum, urine and peritoneal/pleural effusion via UHPLC–MS/MS applicable to personalized antibiotic dosing research

paper_content:

Abstract Management of the therapy of life-threatening bacterial infection is extremely based on an optimal antibiotic treatment. Achieving the correct vancomycin dosage in blood and target tissues can be complicated in special situations, e.g., where large fluid sequestration and/or acute renal failure occur. A UHPLC–MS/MS method operating in electrospray (ESI) positive ion mode was applied for the determination of vancomycin in serum, urine and peritoneal/pleural effusion. Sample pretreatment was composed of dilution and simple protein precipitation where only a small volume (50 μL) of serum, urine or peritoneal/pleural effusion was required. The separation of vancomycin was performed on a Meteoric Core C18 BIO column (100 × 4.6 mm, 2.7 μm) by gradient elution with 0.1% formic acid in water and acetonitrile. The total time of analysis was 4.5 min. The method was found to be linear in the range of 2–60 μM (or 0.5–10 μM) for serum, 0.27–10 μM (or 2–60 μM) for peritoneal/pleural effusion and 25–300 μM for urine, which was adequate for the determination of vancomycin in patient samples. The intra- and inter-day precision was below 8% RSD, and accuracy was from 89 to 104%. The UHPLC/MS–MS method offers a fast and reliable approach to determine vancomycin concentrations in three different human body fluid samples (serum, urine and peritoneal/pleural effusion) with a simple sample pretreatment that was the same for all selected specimens. This method should be applicable to large sample series in clinical (pharmacokinetic/pharmacodynamic) studies.
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paper_title: Determination of the free and total concentrations of vancomycin by two-dimensional liquid chromatography and its application in elderly patients.

paper_content:

Abstract A robust two-dimensional liquid chromatography (2D-LC) method for determining the free and total concentrations of vancomycin in plasma was developed and validated. The 2D-LC system, which exhibited a strong capacity for inhibiting interference, comprised a unique RP1–IEX–RP2 column system and an “Assistant Flow” configuration. Ultrafiltration technology was employed to separate free vancomycin from the protein-bound fraction in human plasma. The influence of ultrafiltration conditions on the free vancomycin concentration was evaluated. The calibration curve was linear over the 0.195–49.92 μg/ml range for the free and total vancomycin concentrations. The within- and between-run precision ranges were 1.5–3.9% and 2.0–4.7% for the total concentration, 1.4–3.3% and 2.4–4.0% for the free concentration, respectively. Ultrafiltration was susceptible to variations in the experimental conditions, including the centrifugation time, the centrifugal force, and the nominal molecular weight limit of the ultrafiltration membrane. A total of 101 serum samples from 84 elderly patients were analyzed by this method. The free vancomycin concentration was 5.88 ± 3.75 μg/ml (range: 0.240–16.79 μg/ml), the total concentration was 12.36 ± 5.36 μg/ml (range: 2.16–27.14 μg/ml), and the unbound fraction was 45.6 ± 18.8% (range: 11.1–96.9%). There was a poor correlation between the free and total vancomycin concentrations (R2 = 0.596, p
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paper_title: New liquid chromatography-tandem mass spectrometry method for routine TDM of vancomycin in patients with both normal and impaired renal functions and comparison with results of polarization fluoroimmunoassay in light of varying creatinine concentrations

paper_content:

Abstract A new LC-MS/MS method with simple sample extraction and a relatively short period of vancomycin analysis for routine therapeutic drug monitoring was developed and validated. 50 μL serum was precipitated using 20 μL 33% trichloroacetic acid and 0.5 mol/L NH4OH was added to increase pH before analysis. A RP BEH C18, 1.7 μm, 2.1 × 50 mm column maintained at 30 °C and tobramycin as internal standard were used. Mass detection was performed in positive electrospray mode. The results obtained with LC-MS/MS method were correlated with an FPIA assay (Abbott AxSYM) using mouse monoclonal antibody. Subjects were divided into three groups according to creatinine levels (53.5 ± 19.1, 150.2 ± 48.4, 471.7 ± 124.7 μmol/L) and Passing-Bablok regression analysis and Bland-Altman analysis were used to compare vancomycin concentrations. The results of subjects with both normal and higher creatinine levels correlated very well and the linear regression model equations were near ideal (LC-MSVAN = 0.947 × AbbottVAN + 0.192 and LC-MSVAN = 0.973 × AbbottVAN − 0.411 respectively). Dialyzed patients with the highest creatinine levels showed about 14% greater vancomycin concentration with the FPIA assay (LC-MSVAN = 0.866 × AbbottVAN + 2.127). This overestimation probably due to the presence of the metabolite CDP ought not to be of clinical relevance owing to the wide range of recommended vancomycin concentration.
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paper_title: Development and validation of a new ultra-performance liquid chromatographic method for vancomycin assay in serum and its application to therapeutic drug monitoring.

paper_content:

OBJECTIVE ::: The aim of this study was to develop and validate an ultra-performance liquid chromatographic (UPLC) method with photodiode array detector for the measurement of vancomycin in human serum samples for therapeutic drug monitoring or other applications. ::: ::: ::: METHODS ::: The method included the extraction of vancomycin in serum by deproteinization with acetonitrile. The analyses were carried out using an ACQUITY UPLC BEH C(18) column (2.1 × 50 mm, 1.7 μm) using acetonitrile and 0.005 M KH(2)PO(4) buffer (pH 2.5) as the mobile phase at a flow rate of 0.3 mL/min, with photodiode array detection at 230 nm. The method was validated for extraction recovery, inter- and intraday precision (relative standard deviation, RSD%), and accuracy and stability of vancomycin in serum. Both the established UPLC method and fluorescence polarization immunoassay (FPIA) were used to measure the prepared quality control (QC) samples (1.0, 7.0, 35.0, 75.0 mg/L) to validate the accuracy of UPLC. Furthermore, both methods were subsequently used to assay the vancomycin concentration in 172 clinical serum samples collected from patients receiving vancomycin in the hospitals localized in Shanghai (China) and 32 control samples from United Kingdom National External Quality Assessment Service (UK NEQAS). ::: ::: ::: RESULTS ::: The retention time of vancomycin was 2.6 minutes. The calibration curve for UPLC was linear over the range 1.0-100.0 mg/L (R(2) > 0.999). The method was fully validated in terms of recovery, selectivity, accuracy, precision, and various conditions. The absolute difference% and RSD% of the prepared QC samples assayed by UPLC were all better than the results by FPIA. A paired t test of the results of the prepared QC samples indicated that the results of all the QC samples had significant difference (P < 0.05), except for the 7.0 mg/L QC samples, which suggested that UPLC was more accurate for the samples containing low or high concentration of vancomycin. A correlation with the Deming model provided a good linear relation between the results of the 2 methods applied to 172 samples, with equation of UPLC = 0.99 × FPIA - 0.19 (R(2)= 0.923), and the agreement of the 2 methods was illustrated using Bland-Altman plot with a mean difference (UPLC - FPIA) of -0.428 mg/L and 95% confidence interval of -8.33 to 7.47 mg/L, respectively. A Student t test comparing results obtained by the UPLC method and group mean results of control samples from UK NEQAS were not significant (P = 0.057). ::: ::: ::: CONCLUSIONS ::: A short analysis time, small amount of serum needed, high specificity, and accuracy make the UPLC method developed in this study appropriate and practical for vancomycin therapeutic drug monitoring and could be applied to other nonserum applications or where requiring superior validation parameters such as for pharmacokinetic/pharmacodynamic studies.
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paper_title: Mass Spectrometry: Principles and Applications

paper_content:

Preface. Introduction. Ion Sources. Mass Analyzers. Tandem Mass Spectrometry (MS/MS). Mass Spectrometry/Chromatography Coupling. Analytical Informaiton. Fragmentation Reactions. Analysis of Biomolecules. Exercises. Appendices. Index.
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paper_title: An ultra-high pressure liquid chromatography-tandem mass spectrometry method for the quantification of teicoplanin in plasma of neonates.

paper_content:

The development and validation of an ultra-high pressure liquid chromatography (UHPLC) tandem mass spectrometry (MS/MS) method was performed with the aim to be applied for the quantification of plasma teicoplanin concentrations in neonates. Pharmacokinetic data of teicoplanin in the neonatal population is very limited, therefore, a sensitive and reliable method for the determination of all isoforms of teicoplanin applied in a low volume of sample is of real importance. Teicoplanin main components were extracted by a simple acetonitrile precipitation step and analysed on a C18 chromatographic column by a triple quadrupole MS with electrospray ionization. The method provides quantitative data over a linear range of 25-6400ng/mL with LOD 8.5ng/mL and LOQ 25ng/mL for total teicoplanin. The method was applied in plasma samples from neonates to support pharmacokinetic data and proved to be a reliable and fast method for the quantification of teicoplanin concentration levels in plasma of infants during therapy in Intensive Care Unit.
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paper_title: Simultaneous analysis of antibiotics in biological samples by ultra high performance liquid chromatography–tandem mass spectrometry

paper_content:

Abstract A rapid and reliable multiclass method was developed for the simultaneous analysis of 21 antibiotics (beta-lactams, aminoglycosides, penicillins, cephalosporins, carbapenems or quinolones) in urine, serum, cerebrospinal fluid (CSF) and bronchial aspirations by ultra-high performance liquid chromatography coupled to tandem mass spectrometry (UHPLC–MS/MS). Prior to chromatographic determination, the analytes were extracted from human biological fluids by simple sample treatments, which imply dilution, liquefaction, or protein precipitation. Several chromatographic conditions were optimized in order to obtain a fast separation (
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paper_title: High-throughput determination of vancomycin in human plasma by a cost-effective system of two-dimensional liquid chromatography.

paper_content:

Therapeutic drug monitoring (TDM) is one of the most important services of clinical laboratories. Two main techniques are commonly used: the immunoassay and chromatography method. We have developed a cost-effective system of two-dimensional liquid chromatography with ultraviolet detection (2D-LC-UV) for high-throughput determination of vancomycin in human plasma that combines the automation and low start-up costs of the immunoassay with the high selectivity and sensitivity of the liquid chromatography coupled with mass spectrometric detection without incurring their disadvantages, achieving high cost-effectiveness. This 2D-LC system offers a large volume injection to provide sufficient sensitivity and uses simulated gradient peak compression technology to control peak broadening and to improve peak shape. A middle column was added to reduce the analysis cycle time and make it suitable for high-throughput routine clinical assays. The analysis cycle time was 4min and the peak width was 0.8min. Compared with other chromatographic methods that have been developed, the analysis cycle time and peak width for vancomycin was reduced significantly. The lower limit of quantification was 0.20μg/mL for vancomycin, which is the same as certain LC-MS/MS methods that have been recently developed and validated. The method is rapid, automated, and low-cost and has high selectivity and sensitivity for the quantification of vancomycin in human plasma, thus making it well-suited for use in hospital clinical laboratories.
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paper_title: A simple quantitative method analysing amikacin, gentamicin, and vancomycin levels in human newborn plasma using ion-pair liquid chromatography/tandem mass spectrometry and its applicability to a clinical study.

paper_content:

Neuroprotective controlled therapeutic hypothermia is the standard of care for newborns suffering perinatal asphyxia. Antibiotic drugs, such as amikacin, gentamicin, and vancomycin are frequently administered during controlled hypothermia, which possibly alters their pharmacokinetic (PK) and pharmacodynamic (PD) profiles. In order to examine this effect an LC-MS/MS method for the simultaneous quantification of amikacin, the major gentamicin components (gentamicin C, C1a and C2), and vancomycin in plasma was developed. In 25μL plasma proteins were precipitated with trichloroacetic acid (TCA) and detection of the components was achieved using ion-pair reversed phase chromatography coupled with electrospray ionization tandem mass spectrometry. The chromatographic runtime was 7.5min per sample. Calibration standards were prepared over a range of 0.3-50mgL(-1) for amikacin and gentamicin and 1.0-100mgL(-1) for vancomycin. At LLOQ accuracy was between 103 and 120% and imprecision was less than 19%. For concentrations above LLOQ accuracy ranged from 98% to 102% and imprecision was less than 6%. Process efficiency, ionization efficiency, and recovery were acceptable. Samples and stock solutions were stable during the time periods and at the different temperatures examined. The applicability of the method was shown by analysing plasma samples from 3 neonatal patients. The developed method allows accurate and precise simultaneous quantification of amikacin, gentamicin, and vancomycin in a small volume (25μL) of plasma.
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paper_title: Accuracy of the analysis of free vancomycin concentration by ultrafiltration in various disease states

paper_content:

In the present study, the accuracy of the analysis of free vancomycin (VCM) by ultrafiltration in various disease conditions was assessed. With VCM as a representative drug, we used clinical plasma samples to investigate the effect of plasma conditions on the volume ratio of ultrafiltrate to sample solution (Vu/Vs) and the consequential effect on the measured free drug concentration (fc). Our results demonstrated that plasma conditions had a significant impact on Vu/Vs by centrifugal ultrafiltration (CF-UF). The Vu by CF-UF ranged from 97 μL to 279 μL among different individuals under the same centrifugation conditions. Total protein levels and the osmotic pressure of plasma were the main influence factors of Vu/Vs in disease states. In contrast, the Vu/Vs by hollow fiber centrifugal ultrafiltration (HFCF-UF) was less influenced by plasma conditions. As a consequence, the results of fc determined by HFCF-UF were more accurate than those by CF-UF for patients with different disease states. HFCF-UF displayed great advantages in clinical samples for accurate analysis of fc. It has been successfully applied to monitor free VCM in clinical plasma samples in routine therapeutic drug monitoring (TDM).
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paper_title: Determination of Vancomycin in Human Plasma, Bone and Fat by Liquid Chromatography/Tandem Mass Spectrometry

paper_content:

A liquid chromatography/tandem mass spectrometry (LC-MS/MS) assay was developed for the determination of vancomycin in human plasma, bone and fat tissue. For vancomycin in plasma, sample was treated with methanol to precipitate the proteins. After centrifugation, the supernatant was diluted with water, and then injected into the LC-MS/MS system. For vancomycin in bone and fat, the pulverized bone/fat samples were immersed in phosphate buffered saline pH 7.3 at 4°C overnight. After centrifugation, the supernatant of bone/fat tissue suspension was treated in the same way as the plasma samples. Vancomycin and aminopterin, the internal standard, were resolved on a C18(2) column using gradient elution of 0.05% formic acid and methanol. The two compounds were detected using electrospray ionisation in the positive mode. Standard curves were linear over the concentration range 0.05 to 50 mg/L (r2>0.99) in plasma, bone and fat. Bias was ≤ ± 10% from 0.05 to 50 mg/L, intra- and inter-day coefficients of variation (imprecision) were <10%, and the limit of quantification was 0.05 mg/L. The assay has been used successfully in a clinical study to investigate the regional delivery of vancomycin in bone and fat after prophylactic administration of vancomycin through an intraosseous route or systemic route, during total knee arthroplasty.
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paper_title: Direct injection LC-MS/MS method for the determination of teicoplanin in human plasma.

paper_content:

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 0.5mL/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 1-50mg/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.
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paper_title: HPLC for Pharmaceutical Scientists

paper_content:

PREFACE. CONTRIBUTORS. PART I HPLC THEORY AND PRACTICE. 1 Introduction (Yuri Kazakevich and Rosario LoBrutto). 1.1 Chromatography in the Pharmaceutical World. 1.2 Chromatographic Process. 1.3 Classification. 1.4 History of Discovery and Early Development (1903-1933). 1.5 General Separation Process. 1.6 Types of HPLC. 1.7 HPLC Descriptors (Vr, k, N, etc.). 2 HPLC Theory (Yuri Kazakevich). 2.1 Introduction. 2.2 Basic Chromatographic Descriptors. 2.3 Efficiency. 2.4 Resolution. 2.5 HPLC Retention. 2.6 Retention Mechanism. 2.7 General Column Mass Balance. 2.8 Partitioning Model. 2.9 Adsorption Model. 2.10 Total and Excess Adsorption. 2.11 Mass Balance in Adsorption Model. 2.12 Adsorption of the Eluent Components. 2.13 Void Volume Considerations. 2.14 Thermodynamic Relationships. 2.15 Adsorption-Partitioning Retention Mechanism. 2.16 Secondary Equilibria. 2.17 Gradient Elution Principles. 2.18 Types of Analyte Interactions with the Stationary Phase. 2.19 Conclusion. 3 Stationary Phases (Yuri Kazakevich and Rosario LoBrutto). 3.1 Introduction. 3.2 Type of Packing Material (Porous, Nonporous, Monolithic). 3.3 Base Material (Silica, Zirconia, Alumina, Polymers). 3.4 Geometry. 3.5 Adsorbent Surface Chemistry. 3.6 Surface of Chemically Modified Material. 3.7 Polymer-Based Adsorbents. 3.8 Stationary Phases for Chiral Separations. 3.9 Columns. 4 Reversed-Phase HPLC (Rosario LoBrutto and Yuri Kazakevich). 4.1 Introduction. 4.2 Retention in Reversed-Phase HPLC. 4.3 Stationary Phases for RPLC. 4.4 Mobile Phases for RPLC. 4.5 pH Effect on HPLC Separations. 4.6 Effect of Organic Eluent Composition on Analyte Ionization. 4.7 Synergistic Effect of pH, Organic Eluent, and Temperature on Ionizable Analyte Retention and Selectivity. 4.8 Examples of Applying pH Shift and Analyte pKa Shift Rules. 4.9 Effect of Temperature on Analyte Ionization. 4.10 Ion-Interaction Chromatography. 4.11 Concluding Remarks. 5 Normal-Phase HPLC (Yong Liu and Anant Vailaya). 5.1 Introduction. 5.2 Theory of Retention in Normal-Phase Chromatography. 5.3 Effect of Mobile Phase on Retention. 5.4 Selectivity. 5.5 Applications. 5.6 Conclusions. 6 Size-Exclusion Chromatography (Yuri Kazakevich and Rosario LoBrutto). 6.1 Separation of the Analyte Molecules by Their Size. 6.2 Molecular Size and Molecular Weight. 6.3 Separation Mechanism. 6.4 Calibration. 6.5 Columns. 6.6 Molecular Weight Distribution. 6.7 Effect of Eluent. 6.8 Effect of Temperature. 6.9 Detectors. 6.10 Solving Mass Balance Issues. 6.11 Aqueous SEC Applications. 7 LC/MS: Theory, Instrumentation, and Applications to Small Molecules (Guodong Chen, Li-Kang Zhang, and Birendra N. Pramanik). 7.1 Introduction. 7.2 Ionization Methods and LC/MS Interfaces. 7.3 Mass Analyzers. 7.4 Role of Instrumental Parameters on Ionization Efficiency in LC/MS. 7.5 Effect of Mobile-Phase Composition on Ionization Efficiency in LC/MS. 7.6 MS Interpretation. 7.7 Practical Applications. 7.8 Conclusions. 8 Method Development (Rosario LoBrutto). 8.1 Introduction. 8.2 Types of Methods. 8.3 Defining the Method. 8.4 Method Development Considerations. 8.5 Method Development Approaches. 8.6 Effect of pH on UV Absorbance. 8.7 Analyte pKa-From an Analytical Chemist's Perspective. 8.8 Reversed-Phase Versus Normal-Phase Separations. 8.9 Instrument/System Considerations. 8.10 Column Testing (Stability and Selectivity). 8.11 Concluding Remarks. 9 Method Validation (Rosario LoBrutto and Tarun Patel). 9.1 Introduction. 9.2 Validation Report. 9.3 Revalidation. 9.4 Assignment of Validation Parameters. 9.5 Distinguishing Drug-Related and Non-Drug-Related Degradation Products. 9.6 Concluding Remarks. 10 Computer-Assisted HPLC and Knowledge Management (Yuri Kazakevich, Michael McBrien, and Rosario LoBrutto). 10.1 Introduction. 10.2 Prediction of Retention and Simulation of Profiles. 10.3 Optimization of HPLC Methods. 10.4 Structure-Based Tools. 10.5 Conclusion. PART II HPLC IN THE PHARMACEUTICAL INDUSTRY. 11 The Expanding Role of HPLC in Drug Discovery (Daniel B. Kassel). 11.1 Introduction. 11.2 Applications of HPLC/MS for Protein Identification and Characterization. 11.3 Applications of HPLC/MS/MS in Support of Protein Chemistry. 11.4 Applications of HPLC/MS/MS in Support of Assay Development and Screening. 11.5 Sources of Compounds for Biological Screening. 11.6 HPLC/MS Analysis to Support Compound Characterization. 11.8 Higher-Throughput Purification Strategies. 11.9 ADME Applications. 11.10 Fast Serial ADME Analyses Incorporating LC-MS and LC-MS/MS. 11.11 Parallel Approaches to Speeding ADME Analyses. 11.12 Automated "Intelligent" Metabolic Stability and Metabolite ID. 11.13 Conclusions. 12 Role of HPLC in Preformulation (Irina Kazakevich). 12.1 Introduction. 12.2 Initial Physicochemical Characterization (Discovery Support). 12.3 Chemical Stability. 12.4 Salt Selection. 12.5 Polymorphism. 12.6 Preformulation Late Stage (Development Support). 12.7 Conclusions. 13 The Role of Liquid Chromatography-Mass Spectrometry in Pharmacokinetics and Drug Metabolism (Ray Bakhtiar, Tapan K. Majumdar, and Francis L. S. Tse). 13.1 Introduction. 13.3 Tandem-Mass Spectrometry (MS/MS). 13.4 Sample Preparation Using an Off-Line Approach. 13.5 Automated Sample Transfer. 13.6 Sample Processing Using an On-Line Approach. 13.7 Matrix Effect and Ion Suppression. 13.8 Regulatory Requirements for LC/MS Method Validation. 13.9 Ritalin(r): An Application of Enantioselective LC-MS/MS. 13.10 GleevecTM (STI571). 13.11 Biomarkers. 13.12 Conclusions. 14 Role of HPLC in Process Development (Richard Thompson and Rosario LoBrutto). 14.1 Responsibilities of the Analytical Chemist During Process Development. 14.2 HPLC Separation Modes. 14.3 Sample Preparation. 14.4 HPLC Detectors. 14.5 Method Development. 14.6 In-Process Monitoring. 14.7 Impurity Identification. 14.8 Establishment of HPLC Selectivity by Stress Studies. 14.9 HPLC Method Validation. 14.10 Technology Transfer. 14.11 Concluding Remarks. 15 Role of HPLC During Formulation Development (Tarun S. Patel and Rosario LoBrutto). 15.1 Introduction. 15.2 Prerequisite for Analytical Chemists During Formulation Development. 15.3 Properties of Drug Substance. 15.4 Properties of Excipients. 15.5 Impact of Excipients on Degradation of API(s). 15.6 Test Methods for Most Common Dosage Forms in which HPLC Is the Primary Technique. 15.7 Forced Decomposition. 15.8 Compatibility of Excipients with API(s) (Type and Ratio). 15.9 Mass Balance. 15.10 Summary of Assay and Related Substances. 15.11 Uniformity of Dosage Units. 15.12 Blend Uniformity (BU). 15.13 Cleaning Verification. 15.14 Extractables/Leachables. 15.15 Dissolution. 15.16 Method Development. 15.17 Method Validation. 15.18 Testing of Samples. 15.19 Automation Opportunities. 15.20 Implementation of Alternative Technologies. 15.21 Challenges and Future Trends. A15.1 Addendum (Common Functional Groups). A15.1.1 Carbonyls. A15.1.2 Nitrogen Functional Groups. A15.1.3 Ethers, Thioethers. A15.1.4 Alkyl/Aryl Halides. A15.1.5 Hydroxyls. A15.1.6 Thiols. A15.1.7 Phenols. A15.1.8 Olefins. A15.1.9 Dimerization. A15.1.10 Ring Transformations. 16 The Role of HPLC in Technical Transfer and Manufacturing (Joseph Etse). 16.1 Introduction. 16.2 Prerequisites for Transfer of HPLC Methods. 16.3 Types of Technical Transfer. 16.4 Different Approaches for Technical Transfer and Manufacturing. 16.5 Potential Pitfalls During Technical Transfer and Manufacturing. 16.6 Conclusion. PART III HYPHENATED TECHNIQUES AND SPECIALIZED HPLC SEPARATIONS. 17 Development of Fast HPLC Methods (Anton D. Jerkovich and Richard V. Vivilecchia). 17.1 Introduction. 17.2 Basic Theory. 17.3 Monolithic Columns. 17.4 Ultra-High-Pressure Liquid Chromatography. 17.5 Separations on Chips. 17.6 Optimizing Gradient Separations for Speed. 17.7 Instrumental Requirements for Operating High-Efficiency Columns. 17.8 Conclusions. 18 Temperature as a Variable in Pharmaceutical Applications (Roger M. Smith). 18.1 The Influence of Temperature on Chromatography. 18.2 Effects on Method Transferability and Reproducibility. 18.3 Elevated Temperature and Pharmaceutical Separations. 18.4 Superheated Water Chromatography. 18.6 Subambient Separations. 18.7 Conclusion. 19 LC/MS Analysis of Proteins and Peptides in Drug Discovery (Guodong Chen, Yan-Hui Liu, and Birendra N. Pramanik). 19.1 Introduction. 19.2 General Strategies for Analysis of Proteins/Peptides. 19.3 Applications for Biotechnology Products and Drug Targets. 19.4 Conclusions. 20 LC-NMR Overview and Pharmaceutical Applications (Maria Victoria Silva Elipe). 20.1 Introduction. 20.2 Historical Background of NMR. 20.3 LC-NMR. 20.4 LC-MS-NMR (or LC-NMR-MS or LC-NMR/MS). 20.5 Conclusions. 21 Trends in Preparative HPLC (Ernst Kuesters). 21.1 Introduction. 21.2 Method Development in Preparative HPLC. 21.3 Columns and Stationary Phases. 21.4 Choice of Preparative LC Technology. 21.5 Detection Tools. 21.6 Conclusion. 22 Chiral Separations (Nelu Grinberg, Thomas Burakowski, and Apryll M. Stalcup). 22.1 Introduction. 22.2 Separation of Enantiomers Through the Formation of Diastereomers. 22.3 Molecular Interactions. 22.3.5 Charge Transfer. 22.4 Mixed Types of Interaction. 22.5 Ligand Exchange. 22.6 Chiral Mobile Phases. 22.7 Method Development for Chiral Separation. 22.8 Concluding Remarks. CHEMICAL AND DRUG COMPOUND INDEX. SUBJECT INDEX.
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Title: Overview of the analytical methods for vancomycin and/or teicoplanin determination in biological matrices

Section 1: INTRODUCTION
Description 1: Introduce the significance of multidrug-resistant (MDR) bacterial infections and the role of glycopeptide antibiotics, vancomycin and teicoplanin, in their treatment. Discuss their mechanism of action, potential toxicities, and the necessity for therapeutic drug monitoring. 

Section 2: Analytical methods
Description 2: Discuss different analytical methods for determining the concentration of vancomycin and/or teicoplanin in various biological matrices, with a focus on methods developed or improved from 2014-2017.

Section 3: Protein precipitation
Description 3: Describe protein precipitation as a method for sample preparation, its effectiveness, and its limitations. Discuss different precipitation agents and their impact on extraction recovery and purity.

Section 4: Solid-phase extraction (SPE)
Description 4: Discuss SPE as a method for sample preparation, its advantages, and its limited application for vancomycin and/or teicoplanin determination in recent years.

Section 5: Direct injection
Description 5: Outline the direct injection method for sample preparation and its recent applications, especially for plasma samples, while highlighting its cost-effectiveness and drawbacks.

Section 6: Ultrafiltration
Description 6: Explain ultrafiltration as a method to separate free and protein-bound drugs, specifically vancomycin, and its potential application despite handling complexity.

Section 7: Liquid-liquid extraction (LLE)
Description 7: Discuss the historical use and recent decline of LLE for vancomycin and/or teicoplanin determination due to the use of toxic solvents and automation difficulties.

Section 8: Chromatographic methods
Description 8: Provide an overview of various chromatographic techniques, particularly LC-MS/MS, used for vancomycin and/or teicoplanin determination, including details on stationary phases, mobile phase compositions, and detection modes.

Section 9: CONCLUSION
Description 9: Summarize the review, emphasizing the advantages and limitations of different sample preparation procedures and chromatographic methods. Highlight the increasing use of LC-MS/MS in clinical practice.

Section 10: Declaration of conflicting interests
Description 10: Disclose any financial and personal relationships that might influence the work.