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Cheng, X.;  Ma, J.;  Su, J. Analytical Methodologies for Determination of Vancomycin. Encyclopedia. Available online: https://encyclopedia.pub/entry/32302 (accessed on 18 May 2024).
Cheng X,  Ma J,  Su J. Analytical Methodologies for Determination of Vancomycin. Encyclopedia. Available at: https://encyclopedia.pub/entry/32302. Accessed May 18, 2024.
Cheng, Xin, Jingxin Ma, Jianrong Su. "Analytical Methodologies for Determination of Vancomycin" Encyclopedia, https://encyclopedia.pub/entry/32302 (accessed May 18, 2024).
Cheng, X.,  Ma, J., & Su, J. (2022, November 01). Analytical Methodologies for Determination of Vancomycin. In Encyclopedia. https://encyclopedia.pub/entry/32302
Cheng, Xin, et al. "Analytical Methodologies for Determination of Vancomycin." Encyclopedia. Web. 01 November, 2022.
Analytical Methodologies for Determination of Vancomycin
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Vancomycin is regarded as the last resort of defense for a wide range of infections due to drug resistance and toxicity. The detection of vancomycin in plasma has always aroused particular concern because the performance of the assay affects the clinical treatment outcome. With the update of technology, bioassay, immunoassay, LC appreared in sequence with respective characteristic. 

vancomycin analytical method human plasma bioassay

1. Introduction

Vancomycin is a tricyclic glycopeptide antibiotic produced by the fermentation of Streptomyces orientalis that was isolated in soil samples from the jungles of Borneo, Indonesia in 1956 [1]. It inhibits bacterial synthesis by three main mechanisms: inhibition of the synthesis of peptidoglycan, alteration of the cell membrane permeability, and interference with RNA synthesis in the cytoplasm [2]. The antibacterial spectrum of vancomycin mainly includes aerobic Gram-positive bacteria such as Staphylococcus, Streptococcus, Enterococcus, Corynebacterium, Listeria, and Clostridium difficile [1]. Currently, vancomycin is primarily used for the treatment of infections due to methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-resistant Staphylococcus epidermidis (MRSE), pseudomembranous enteritis due to Clostridium difficile, an alternative to β-lactam allergy, the prevention of endocarditis, and infections during prosthetic implantation [3].
Vancomycin is poorly absorbed in the digestive tract, and intramuscular injection can cause severe local pain and tissue necrosis. It is routinely administered intravenously via 5% dextrose or 0.9% saline. After injection, vancomycin is rapidly distributed to many body tissues, reaching effective therapeutic concentrations in the lung, heart, synovial fluid, peritoneal fluid, bone, and kidney. However, it cannot cross the blood–brain barrier in a non-inflammatory state. Its protein binding rate is about 55%. Moreover, in patients with normal renal function, more than 90% of the drug is excreted in the unchanged form via the kidneys [1][4]. Its pharmacokinetics is influenced by several factors including the patient’s age, body weight, serum albumin, urine pH, and combined medications [5]. The half-life of vancomycin is closely related to renal function, ranging from 6 h in those with normal renal function to 7 days in anuric patients [1].
Vancomycin is a time and concentration-dependent (AUC-dependent) antibiotic with a post-antibiotic effect. According to pharmacokinetic (PK)/pharmacodynamic (PD) theory, the evaluation indicator for therapeutic drug monitoring (TDM) of vancomycin is the area under the concentration–time curve (AUC)/minimum inhibitory concentration (MIC) ratio, while with a AUC/MIC ratio ≥ 400 (based on MIC ≤ 1 µg mL−1), vancomycin can achieve a clinical effect [4][6]. However, when MIC > 1 µg mL−1, it is recommended to switch to another drug [6]. Vancomycin has a narrow therapeutic window (effective concentration is close to toxic concentration). Insufficient drug concentration can easily lead to the development of bacterial resistance, and too high a concentration is prone to serious adverse effects on the body [7] such as nephrotoxicity, ototoxicity, hypotension, phlebitis, hypersensitivity reactions, red man syndrome, neutropenia, chills, fever, and interstitial nephritis. Therefore, it is necessary to perform a TDM for vancomycin.

2. Bioassay

Bioassays provide a more visual assessment of vancomycin concentrations based on the antibacterial activity of vancomycin in vitro. Bioassay methods for vancomycin were studied mainly in the 1980s (Table 1) [8][9][10][11][12][13]. The basic steps are as follows: uniform inoculation of an indicator bacterium on a suitable medium, punching holes in the medium with a punch and pouring in vancomycin solution [14], or soaking paper sheets in vancomycin-containing solution and sticking them on the medium [9][10][11][12][13]. The inhibition circle diameter is linearly related to the vancomycin concentration or its logarithm. The corresponding vancomycin concentration can be calculated from the standard curve and the inhibition circle diameter. The key in this method is the selection and preparation of the indicator bacterium and medium. Bacillus spp. is sensitive to most antibiotics and is often used as an indicator bacterium. Furthermore, the medium should neither affect the indicator bacterium’s growth nor the antibiotic activity [15]. Other factors such as the pore size, incubation time, and incubation temperature can also affect the diameter of the antibacterial coil. This method’s standard concentration range (0.8–80 µg mL−1) covers the routine dose concentration range of vancomycin, which meets the clinical needs. Moreover, it is cheaper compared to immunoassays and liquid chromatography. The bioassay results were consistent with those of FPIA, HPLC, RIA, and fluorescence immunoassay [8][12]. Nevertheless, the operational steps are more cumbersome than other methods such as immunoassay and overnight incubation, which further prolong the experimental operation time. Although investigators often repeat a single test ten times [8] or four times [9] in trials to avoid random error, the precision and accuracy are still not better than other methods. In addition, patients are often not mono-medicated in clinical settings. It is true that aminoglycosides can be inhibited by increasing the concentration of NaCl to 6.0% in the plate, and rifampicin does not affect the inhibitory activity of vancomycin. However, other drugs such as β-lactams, macrolides, and sulfonamides still affect the application of this method [8][11].
Table 1. Bioassay for vancomycin.
In conclusion, the method is limited in its clinical application due to the use of multiple drugs in clinical practice and the long operation time. Nevertheless, in primary hospitals without expensive instruments (e.g., immunoassay analyzer, liquid chromatograph), a bioassay is still recommended for assaying the drug concentration in plasma. However, further research is needed to inactivate the activity of other antibacterial drugs to reduce the interference of vancomycin detection.

3. Immunoassay

Vancomycin has a molecular weight of 1449 Da, which does not stimulate the human body to produce relevant antibodies. Therefore, an immunoassay is not affected by anti-vancomycin antibodies produced by the human body [16]. Due to its small molecular weight, the competition method is the primary type of immunoassay for vancomycin. This method uses labeled vancomycin and free vancomycin in blood to compete for binding to the corresponding antibody; the more free-vancomycin is present in the blood, the less labeled vancomycin can bind to the antibody. The markers can be isotopes, fluorescence, or enzymes. According to the type of marker, these methods can be classified as the RIA, fluorescent immunoassay (FIA), and enzyme immunoassay (EIA).

3.1. RIA

Crossley et al. [13] and Fong et al. [16] detailed the process of the vancomycin radioimmunoassay. First, vancomycin is conjugated with bovine serum albumin and then injected intravenously into test rabbits to obtain antibodies; 3H acrylated or 125I iodinated vancomycin, respectively; the vancomycin standard solution or vancomycin in serum competed with the labeled vancomycin to bind antibodies, and the radioactive signal was recorded by scintillation spectrometry (3H) or γ-counter (125I). The method’s sensitivity was 0.04 ng mL−1 to the maximum, while that of the biological method was 0.8 µg mL−1. RIA was significantly more sensitive than the biological method. Moreover, vancomycin could be detected in the serum and urine after oral vancomycin administration, which has an advantage in terms of small patient sample size or studying vancomycin metabolism in the organism [16]. Moreover, the results corresponded well with those obtained by the biological methods, FPIA and HPLC, while it was slightly less accurate and precise [13][14]. The RIA results were high compared to the biological method. Some studies attributed this to the degradation of vancomycin at low pH [13]. Meanwhile, the isotope quenching should not be neglected in the experiment. Disadvantages of RIA are apparent [17]: preservation and waste disposal of radioisotope reagents; hazards to humans; relatively short shelf-life; the need for dilution before sample detection; the need to separate antibody-bound and free fractions before counting; and the fact that counters are expensive and not equipped in routine bacterial laboratories. Based on the above facts, RIA has not been routinely used in laboratories.

3.2. FIA

FPIA is the most widely used method in FIA. The method requires a fluorescence polarizer and a fluorescently labeled antigen as a tracer. Meanwhile, it needs to avoid interference from endogenous fluorescence [18]. The relevant principles were elucidated in a previous literature review [18][19]. The tracer competes with the analyte to bind the antibody, and the tracer binds the antibody with a significantly higher fluorescence polarization value than the free tracer. Eventually, the analyte concentration is inversely related to the detected fluorescence polarization value. Schwenzer et al. [20] first reported the measurement of vancomycin by the FPIA instrumental method (Abbott TDx) in 1983. Backes [21] et al. used HPLC to confirm that the presence of the crystalline degradation product (CDP-1) was responsible for the high FPIA results. CDP-1 has two isomers, CDP-1-M (major) and CDP-1-m (minor), both of which have no antibacterial activity but can cross-immunoreactive with anti-vancomycin antibodies, thus leading to high results, especially in patients with kidney injury [21][22]. Other factors such as the poor stability of standards [23], drug accumulation due to altered vancomycin pharmacokinetics in patients with kidney injury [24], and the use of sheep-derived polyclonal antibodies in the FPIA method [25] may contribute to the high FPIA results. For clinical decision-making, the results of FPIA were elevated by approximately 14%. However, there was no need to adjust the vancomycin treatment dosage, given that the results were still within the vancomycin treatment window [26]. In conclusion, as a quick and easy method for the laboratory detection of vancomycin blood levels, the method’s accuracy can be improved by increasing the frequency of the assay, the use of monoclonal antibodies, and proper preservation of the standards and drugs. Other methods are recommended for patients with renal injuries such as EMIT and HPLC.

3.3. EIA

EMIT is one of the more commonly used clinical methods in EIA and can be used to test samples using conventional biochemical analyzers and commercial kits rapidly. It is performed by competing for the enzyme-labeled semi-antigen with the analyte to bind the antibody. The activity of the enzyme-labeled semi-antigen will be lost after binding to the antibody. Finally, the analyte concentration will be determined based on the change in absorbance after the enzyme-catalyzed reaction [27][28]. EMIT and FPIA, commonly used in clinical laboratories, are often used for comparison by investigators. They correlated well with each other. Both had fine precision and accuracy, which can meet the needs of clinical laboratories [22][23][24][25]. However, a multicenter retrospective study from the United Kingdom showed that EMIT would be more prone to random errors occurring than FPIA [29]. FPIA is susceptible to high results due to CDP-1, as mentioned previously. Both methods are more straightforward and faster than other methods such as the bioassay and HPLC, and they are not as harmful as RIA, so they are widely used in clinical laboratories. Both require regular internal quality control and external quality assessment to ensure the precision and accuracy.
It should be noted that EIA is occasionally affected by endogenous cross-reactive substances such as rheumatoid factor, heterophilic antibodies, paraproteins, C-reactive protein, or unexplained substances affecting enzyme activity, leading to falsely elevated vancomycin measurements and treatment failure due to the underdosing of patients [30][31]. Laboratory workers can reduce the interference of other protein-like substances by polyethylene glycol precipitation or heat inactivation methods. Alternatively, they can further use HPLC or LC-MS/MS to detect vancomycin concentrations. When clinicians encounter results that are inconsistent with clinical outcomes, they should consider the presence of assay influencing factors and communicate with laboratory workers promptly to minimize the impact of false results on the patients’ treatment decisions.

4. LC

LC is an essential method for isolating and detecting vancomycin in plasma or serum.

4.1. Pretreatment

Drugs in blood exist in both bound and unbound forms by binding proteins or not. The unbound form (free form) in blood has antibacterial activity and free drug can be separated from the bound drug by certain means such as equilibrium dialysis, ultrafiltration, ultracentrifugation, on-line or off-line methods, and non-separative methods, as previously reviewed [32]. Protein-binding studies have used these methods to investigate the relationship between free and total vancomycin in plasma, aiming at exploring whether total vancomycin can be used to assess free vancomycin [33][34]. Unfortunately, both of the results were negative. In addition, the experimental conditions (molecular weight cut-off, centrifugal force and time, pH, temperature) may affect the isolation of free vancomycin by ultrafiltration [35]. However, studies on free vancomycin remain scarce despite its importance. Total vancomycin is routinely tested in the laboratory [33]. In the LC method, pretreatment is conducted to eliminate interference from plasma proteins and other macromolecular substances. All samples were physically separated by centrifugation, and most studies added precipitates to denature the protein [26][36][37][38][39][40][41][42][43][44][45][46][47][48][49][50][51][52][53][54][55][56][57][58][59][60][61][62][63]. In addition, a few studies isolated the proteins in a combined solid phase extraction (SPE) [36][58] manner. Precipitants include ACN, MeOH, TCA, and HClO4; the most commonly used are ACN and MeOH solutions. ACN and MeOH are often used as subsequent mobile phase components. Hence, neither increases the interference analyzed in chromatography, further reducing the matrix effect. There was only one recovery result for TCA and HClO4 as precipitants. Bijleveld et al. found a maximum recovery of 70% at 15–35% (w/v) TCA concentration. Combined with other extraction methods, the analyte could be purified efficiently. For example, SPE effectively separated analytes and interferents, even though vancomycin 0.05 µg mL−1 recovery could reach 87.6% [58]. The complex sample pretreatment process improves the purity of the analytes. However, it also increases the labor and economic costs, which is not conducive to detecting drug concentration. Aqueous ACN or MeOH solution for protein precipitation, then centrifugation, followed by supernatant extraction, is the most economical and convenient sample pretreatment mode. After sample pretreatment, the supernatant is evaporated by liquid nitrogen to a dry powder state and can be used for the next chromatographic step.

4.2. Internal Standard (IS)

Appropriate IS allows for monitoring of the sample pretreatment process, column injection volume, and even the evaluation of the calculated sample volumes. The amount of the target analyte can be assessed or calculated from the peak area of the IS and the peak area of the analyte. Although a number of studies [35][39][41][44][46][48][49][64] have not mentioned or used IS, these studies still obtained reliable findings. Most studies considered the inclusion of IS in the sample pretreatment. For the selection of IS, ultraviolet (UV) detection and mass spectrometry (MS) detection have different requirements. In UV detection, the peak overlap between the analyte, IS, and endogenous plasma should be avoided. The λmax of IS should be as close as possible to the λmax of the analyte and has good absorbance [37]. Hence, tinidazole [36], acetaminophen [37], 3-nitroaniline [38], zidovudine [65], ristomycin [40], caffeine [42], ketoprofen [43], and cefuroxime [45] can be used as IS for the vancomycin assays. The effect of the matrix ionization of analytes in MS detection could be best compensated using a stable isotope-labeled IS. Therefore, isotopically labeled vancomycin with similar extraction recovery, chromatographic characteristics, and ionization response to the desired analyte are the preferred choice of IS for MS detection for commercialization and the most applied vancomycin derivatives in the studies were vancomycin derivatives such as desmethyl vancomycin [50][51][53], desleucine vancomycin [57], and vancomycin-glycin [60], all of which have similar features to the isotope-labeled vancomycin. Others such as tobramycin [26], PABA [47], erythromycin [66], roxithromycin [52], linezolid [54], 10-hydroxycarbazepine [55], atenolol [58], 13C3-caffeine [59], polymyxin B [61], and kanamycin B [63] were confirmed to be applied as IS in the study.

4.3. Stationary Phase and Mobile Phase

Based on vancomycin polarity and molecular weight size, vancomycin is most often separated using the reversed-phase liquid chromatography mode [26][35][36][37][38][39][40][41][42][43][44][45][46][47][48][49][50][51][52][53][54][55][58][59][60][61][62][64][65][66][67], and a few studies have used hydrophilic interaction chromatography [56][57] and reverse ion exchange chromatography [63]. The difference between the various separation modes lies in the choice of stationary and mobile phases. The most commonly applied stationary phase in the reversed-phase liquid chromatography mode was a C18 column [26][35][37][38][41][42][43][44][45][46][47][48][49][50][51][52][53][54][55][59][60][61][62][64][65][66][67], and a few used the C8 column [36][39][58][64] and aminopropyl silica column [40]. In reversed-phase liquid chromatography mode, the most commonly used mobile phases for UV detection were acetonitrile and pH 2.5 buffer such as phosphate buffer [35][37][38][40][41][42][45][49][64][65], while MS detection was performed with acetonitrile and 0.1% formic acid [50][51][52][53][54][55][58][59][61][62][67]. HILIC hydrophilic interaction columns are hydrophilic and elute in the opposite order to reversed-phase liquid chromatography. This chromatographic mode retains highly polar and hydrophilic drugs well and is highly compatible with electrospray mass spectrometry detection [56]. This mode results in longer vancomycin retention times due to vancomycin polarity and hydrophilic effects. However, in the studies by Parker et al. [56] and Oyaert et al. [57], the vancomycin retention time was 2 min and 2.7 min, which effectively solved the problem of the long retention time of HILIC. In addition, Bijleveld et al. [63] used reverse ion exchange chromatography for vancomycin separation. The most important feature of this study was the addition of ionic pair 200 mM perfluorovaleric acid/130 mM ammonium acetate to the mobile phase, and other conditions were the same as those of reversed-phase liquid chromatography.

4.4. Detection

Various detection methods have been used including UV detection, MS detection, photodiode array (PDA), electrochemical detection (ECD), and fluorescence detection (FLD), with the first two being the most commonly used detection methods. Ghasemiyeh et al. [37] plotted a standard curve of vancomycin concentration versus the ratio of vancomycin peak area to acetaminophen peak area to obtain the vancomycin concentration, while Hu et al. [39] recorded the UV–Vis spectra for each retention time and obtained a matrix (elution time × wavelength) for each sample analyzed, which was mathematically separated using two trilinear decomposition algorithms. Neither of these studies used the detection method described above. The wavelength range in UV detection was 205–282 nm, with 240 nm being the most used detection wavelength [35][41][42][43][44][45][46][49][64][65]. MS detection detectors included triple quadrupole [26][51][52][53][54][56][57][59][60][61][62][63][67], Q-Trap [50][55], and Orbitrap [58], and the ion sources were all in ESI (+) mode. The MS assay (10–200 µL) generally used less sample than the UV assay (50–2000 µL), PDA (200 µL, 1000 µL) and fluorescence assay (500 µL). The amount of plasma was significant for certain special populations. Moreover, the sensitivity and linear range of MS and UV detection was comparable at 0.1–1 µg mL−1 (sensitivity range) and 0.1–100 µg mL−1 (linear range), respectively, except for one study that used the Q-Trap assay with a sensitivity of 1 ng mL−1. In addition, the sensitivities of the FLD and PDA assays could reach 2 ng mL−1 and 1 ng mL−1, respectively.

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