Bioanalysis of aminoglycosides using high-performance liquid chromatography

Aminoglycosides are broad-spectrum antibiotics used in the treatment of gram-negative bacterial infections. Due to their nephrotoxic and ototoxic potential (narrow therapeutic index), the use of aminoglycoside for clinical indications requires monitoring. The objective of this review was to identify relevant literature reporting liquid chromatographic methods for the bioanalysis of aminoglycosides in both preclinical and clinical settings/experiments. Data on liquid chromatographic methods were collected from articles in an online academic database (PubMed, Science Direct, Scopus, and Google Scholar). All 71 articles published from 1977 to 2020 were included in the review. Reversed-phase liquid chromatography was the most used method for the bioanalysis of aminoglycosides. Fluorescence or ultraviolet detection methods were mostly used from 1977 to 2002 (51 articles), while mass spectrometry was predominantly used as a detector from 2003 to 2020 (15 articles). Sixty-seven articles reported calibration ranges, which varied significantly for the various drugs assayed: some in the range of 0.1-0.5 ng/mL and others 1250-200000 ng/mL. Also, 61 articles reported R2 values (0.964-1.0) for almost all analytes under consideration. Sixty-three articles reported percent recoveries mostly between 61.0 % to 114.0 %, with only two articles reporting recoveries of 4.9 % and 36 %. Out of the 71 reviewed articles, 56 reported intermediate precision values ranging between 0.331 % to 19.76 %, which is within the acceptable limit of 20 %. This review will serve as a guide for research and/or routine clinical monitoring of aminoglycosides in biological matrices.


Introduction
Aminoglycosides are broad-spectrum antibiotics that are used in the treatment of gram-negative bacterial infections [1,2]. Aminoglycosides elicit their pharmacological effect by binding to the 16S rRNA ribosomal subunit of bacteria and blocking mRNA translation, altering protein synthesis. Structurally, this class of antibiotics has amino sugars in their core connected via glycosidic linkages to a dibasic aminocyclitol [3]. Aminoglycosides are relatively hydrophilic, hence, rarely undergo biotransformation in vivo. Aminoglycosides, to some extent, bind to plasma proteins and are excreted entirely unchanged in urine [4]. Streptomycin, netilmicin, tobramycin, kanamycin, spectinomycin, gentamicin, neomycin, amikacin, and paromomycin are examples of aminoglycosides [5,6].
Aminoglycosides are known to possess nephrotoxic and ototoxic potentials, limiting their clinical use [2]. In addition, due to their narrow therapeutic index, therapeutic monitoring is required for aminoglycosides. Especially among patients with underlying renal problems, monitoring aminoglycosides in biological matrices ensures optimal therapy and reduces toxicity [7].
There are several validated bioanalytical methods used to quantitatively determine levels of aminoglycosides in biological matrices. Microbiological assays, radioimmunoassay (RIA), radioenzymatic assays, fluorescence polarization immunoassay (FPIA), high-performance liquid chromatography (HPLC), gas chromatography (GC) and mass spectrometric techniques are some common examples [8][9][10][11][12]. Of these methods, HPLC is the most preferred or routinely used [13]. Indeed, several studies have used HPLC for the bioanalysis of aminoglycosides [14][15][16]. Although there are recently published reviews on aminoglycosides, the focus of these reviews have been a) pre-treatment and analysis methods of aminoglycosides in food [17], b) challenges in the development of analytical test procedures for aminoglycosides [18], and c) determination of kanamycin by high-performance liquid chromatography [19].
Although the list of articles used in this review may not be exhaustive, suitable liquid chromatographic conditions used in assaying aminoglycosides in biological matrices have been identified. Performance metrics of the various liquid chromatographic assays have also been appraised. Also, highlights of current procedures, scope, characteristics, and limitations of chromatographic methods used in assaying aminoglycosides in biological matrices have been provided. Although this is not a systematic review, it will serve as a comprehensive reference for subsequent related research that may involve the assay of aminoglycosides.

Methods
This study reviewed relevant and accessible articles on liquid chromatographic assays of aminoglycosides in biological matrices from 1977 to 2020. Articles were retrieved from journals in online academic databases (PubMed, Science Direct, Scopus, and Google Scholar) and limited to only the English

Aminoglycosides
Sixty-seven (67) out of the 71 articles reported the use of HPLC in the bioanalysis of aminoglycosides, while four articles used ultra-performance liquid chromatography (UPLC) [35]. The relevant aspect of liquid chromatographic conditions used in the various articles, such as matrix, sample preparation, flow rate, column selection, mobile phase, and detection, have been summarized (Table 1)

Sample cleanup
The matrix in which a drug is found can affect bioanalysis. Also, matrices can compromise the sensitivity and selectivity of bioanalysis methods [86][87][88][89], which is deemed a "matrix effect". The matrix effect could be due to endogenous or exogenous agents. Some endogenous substances include salts, carbohydrates, amines, urea, lipids, peptides, and metabolites [90,91]. Exogenous substances may include anticoagulants, blood preservatives, and mobile-phase additives such as buffer salts [92,93]. Sample cleanup is required to isolate analyte(s) of interest from a matrix.
Sample cleanup tends to reduce or remove matrix components and concentrate the analyte(s). This process improves assay sensitivity and selectivity. An optimal sample cleanup system should be capable of minimizing matrix effect while ensuring reliable extraction recovery [94]. Basically, there are three sample preparation methods that can be applied during HPLC bioanalysis of aminoglycosides; protein precipitation (PPT), solid-phase extraction (SPE), and liquid-liquid extraction (LLE) [4]. All the articles reviewed provided sample preparation methods; 41 used PPT, 33 SPE, and 4 LLE. One article reported the use of ultrafiltration as a sample preparation method [56], 6 articles reported a combination of PPT and SPE [25,42,46,47,61,62], and 1 article used a combination of PPT, SPE, and LLE [72].
Also, four articles adopted the LLE as a sample preparation technique for the extraction of netilmicin, gentamicin, amikacin, and dihydrostreptomycin. Aminoglycosides are hydrophilic in nature, and LLE may not be ideal for extraction.

Chromatographic conditions
All articles reviewed reported chromatographic conditions, and these are highlighted in this review.

Mode of chromatography
The most-reported mode of chromatography used for bioanalysis of aminoglycosides was reversedphase HPLC (66 articles). This may be due to the polarity of the analytes of interest [96]. Other modes of chromatography for the separation of aminoglycosides used were hydrophilic interaction liquid chromatography (2 articles) for the analysis of amikacin, gentamicin, kanamycin, neomycin, paromomycin, and tobramycin [31,69]; hydrophobic interaction chromatography (1 article) for the analysis of neomycin [14]; and ion-exchange chromatography (1 article) for the analysis of gentamicin [40]. One article reported the use of mixed-mode chromatography using both reversed-phase and normal chromatography for the analysis of amikacin [41].

Mode of elution
Although the mobile phase is not directly responsible for chromatographic separation, it can affect chromatographic resolution, selectivity, and efficiency [96]. The selection of a suitable mobile phase for the HPLC system is dependent on the physicochemical properties of the analyte [98]. Since reversed-phase chromatography was reported by most articles, mobile phase solvents used consisted of an aqueous buffer and a non-ultraviolet active water-miscible organic solvent [96]. The solvents included acetonitrile for the separation of netilmicin, gentamicin, and amikacin [21,39,40,47,60]; methanol and water mixture for the separation of gentamicin, amikacin, tobramycin, netilmicin, dibekacin, and sisomicin [20,22,24,36,44]; and heptanesulphonic acid for separation of gentamicin, and netilmicin [58,59]. Other solvents such as tetrahydrofuran for the separation of amikacin [15]; octanesulphonate for the separation of tobramycin, sisomicin, and netilmicin [48]; and ethylene glycol for the separation of sisomicin [54] were also reported. In the case where ionisable analytes were present, the pH of the mobile phase had a significant effect on the ionization state(s) of the analyte(s), which eventually affected resolution. Thus, the buffer reported by most articles were aqueous, which included tris buffer [23], sodium acetate [24], and potassium phosphate buffer [15]. Basic analytes such as aminoglycosides are protonated at low pH when ionized.
Fluorescence (~50 %) was the most used method for the detection of streptomycin, gentamicin, amikacin, dihydrostreptomycin, kanamycin, netilmicin, isepamicin, sisomicin, neomycin, tobramycin, dibekacin, astromicin, and micronomicin. However, non-fluorescence drugs such as aminoglycosides are mostly difficult to detect by this mode due to the absence of a fluorophore. This shortcoming can be mitigated by derivatization [100]. Additionally, the mobile phase used must be selected with care, as highly polar solvents or halide ions can quench fluorescence. It is noteworthy that fluorescence is mostly preferred to UV detection due to its high sensitivity and selectivity [101]. Aminoglycosides assayed in various studies with fluorescence detectors were achieved at an excitation wavelength range of 220 nm [21] to 490 nm [77] and emission wavelength range of 415 nm [57] to 531 nm [74].
In this review, HPLC coupled with resonance Rayleigh scattering detection was used in analyzing three aminoglycosides; amikacin, netilmicin, and etimicin [32]. An advantage of the resonance Rayleigh scattering detector over other spectroscopic techniques is that the detection limit is lower by several orders of magnitude [104].
The pulsed electrochemical detector is mostly used to analyze carbohydrates and polyalcohol. They are also used in analyzing amines, amino acids, and sulphur-containing compounds [105,106]. One (1) article reported the use of HPLC coupled with the pulsed electrochemical detector in the bioanalysis of amikacin [15]. This approach was used to address the shortfall of using derivatization for the detection of aminoglycosides [15].
Chemiluminescence allows the detection of analytes at ultra-high sensitivity. In this review, 1 article reported the use of chemiluminescence for the detection of amikacin, and the chemiluminescence reagent used was luminol in combination with hydrogen peroxide and Cu 2+ [71].
HPLC coupled with evaporative light scattering detector is rapidly becoming a quasi-universal detector, mitigating the need for derivatization of non-absorbing analytes. In this review, one article reported the use of an evaporative light scattering detector for the direct determination of tobramycin [70].

Performance metrics
Performance metrics are quantifiable terms that indicate the quality of an analytical process. Some performance metrics include specificity, sensitivity, linearity, the lower limit of quantification (LLOQ), limit of detection (LOD), precision, accuracy, and calibration range. In this review, all the 71 articles reported some aspects of method performance characteristics. Method performance characteristics reported by most articles included calibration range, linearity, recovery, repeatability, and intermediate precision ( Table  2). Out of the 71 articles reviewed, only four reported on matrix effect [63,[83][84][85]. Resolution was reported by one article [75].
The quality of a bioanalytical method is highly dependent on the linearity of the calibration curve [108]. The linearity of the calibration curve is usually expressed as a coefficient of correlation (R 2 ). The coefficient of correlation close to 1 (R 2 ≈1) is mostly considered ideal. In the current review, 61 articles reported R 2 values for almost all analytes under consideration, whilst 10 articles did not [23,28,33,39,45,47,50,55,56,84]. The reported coefficient of correlation was between 0.964 [75] and 1.0 [48,60,76,82]. Additionally, y-intercept, the slope of the regression of line, and residual sum of squares can also be used in evaluating linearity [109]. Out of 71 articles reviewed, 39 articles reported values for both the slope of regression and the y-intercept.
Accuracy of a bioanalytical method is normally expressed as the percent recovery by the assay of the known added amount of analyte. In this study, 63 articles reported percent recoveries that ranged mostly between 61.0 % [72] to 114.0 % [5]. Two (2) articles reported recoveries of 4.9 % [28] and 36 % [76], respectively.
LLOQ is defined as the amount of analyte in a biological matrix that can be quantitatively determined with suitable precision and accuracy [110]. Thirty-six (36) articles reported LLOQ for aminoglycosides assayed between 1 ng/mL [72] to 2340 ng/mL [79]. Forty-one (41) articles also reported LOD values in the range 0.3 ng/mL [61] to 75000 ng/ml [56]. There were instances where the same article reported both the LLOQ and LOD.
Repeatability expresses the closeness of results obtained with the same sample using the same procedure, operators, measuring system, operating conditions, and location over a short period of time.                           n.a.

Conclusion and outlook
Despite reported nephrotoxic and ototoxic potentials of aminoglycosides, their use in clinical settings remains relevant. The current study sought to review bioanalytical methods (specifically liquid chromatography) used in the assay of aminoglycosides in biological matrices. In all, 71 articles were reviewed, and 66 of these articles reported the use of reversed-phase liquid chromatography as a bioanalytical method [7,119].
The commonest sample treatment procedures adopted in the analysis of aminoglycosides using HPLC were protein precipitation (50 %) and solid phase extraction (39 %). Surprisingly, none of the current sample preparation methods was used by any of the articles reported in this review. It will be interesting if recent sample preparation methods such as solid-phase microextraction, micro-solid-phase extraction, dispersive micro-solid-phase extraction, magnetic solid-phase extraction, microextraction by packed sorbent, stir bar sorptive extraction, spin column extraction, liquid-phase microextraction, single-drop microextraction, hollow fiber liquid-phase microextraction, dispersive liquid-liquid microextraction, molecularly imprinted solid-phase extraction, and molecularly imprinted solid-phase micro-extraction [120][121][122] could be applied for bioanalysis of aminoglycosides with the potential of improving method sensitivity and selectivity.
Fluorescence (50 %), UV (24 %), and mass spectrometry (21 %) were the most adopted mode of detection in the assay of aminoglycosides, according to this review. Since mass spectrometry has been established as the detection mode of choice for bioanalysis of aminoglycoside using liquid chromatography in recent years, it is strongly recommended for use except in resource-challenged countries where fluorescence or UV detection methods can be applied after derivatization.
There is the need to establish a therapeutic reference range for all the clinically reported 20 aminoglycosides since the calibration range of analytical methods for the bioanalysis of aminoglycosides should cover such a range. It was quite surprising that some of the calibration range was outside the established therapeutic reference range. It is recommended that future liquid chromatography methods for the analysis of aminoglycosides should have calibration ranges covering established reference therapeutic ranges.
Although this review is not a systematic one, the information provided is intended to serve as a comprehensive reference for related research that may involve the assay of aminoglycosides (pharmacokinetic or drug monitoring studies).