Residue analysis of 10 aminoglycoside antibiotics in aquatic products by multiwalled carbon nanotubes combined with mixed-mode ion exchange liquid chromatography-tandem mass spectrometry.

An ultra-high-performance liquid chromatography-tandem mass spectrometry method was developed for simultaneous determination of 10 kinds of aminoglycosides in edible parts of aquatic products. The samples were extracted with 10 mmol/L potassium dihydrogen phosphate buffer solution, then the pH value of the extract was adjusted to neutral by sodium hydroxide. Half volume of the extract was loaded onto multiwalled carbon nanotubes cartridge. All the target compounds were separated on a mixed-mode ion exchange column and detected by ultra-high-performance liquid chromatography-tandem mass spectrometry with electrospray in the positive ionization mode. Under optimized conditions, this method had a good linearity with a squared correlation coefficient > 0.999. For neomycin, the limit of detection and limit of quantification were 5.0 µg/kg and 10.0 µg/kg, respectively; for hygromycin B and apramycin, values were 2.0 µg/kg and 5.0 µg/kg, respectively; for the other seven kinds of aminoglycosides, values were 1.0 µg/kg and 2.0 µg/kg, respectively. The average recoveries presented 75.8%-107.2% with intra- and interday reproducibility ranging between 3.8% and 12.5%. The method was rapid with good separation and sharp peak shapes, had the characteristicsis of high accuracy and good precision, and was suitable for simultaneous determination of 10 kinds of aminoglycosides in aquatic products.

Chemiluminescence microarray immunoassay for multiple aminoglycoside antibiotics based on carbon nanotube-assisted signal amplification.

The simultaneous determination of multiple analytes has been an urgent demand in screening of antibiotic residues in food products of animal origin due to its higher analysis efficiency. Five aminoglycoside antibiotics (AGAs) have been monitored in milk, including gentamicin (GEN), kanamycin (KAN), neomycin (NEO), and streptomycin/dihydrostreptomycin (STR/diSTR). A chemiluminescence microarray immunoassay (CLMIA) based on nitrocellulose membrane had been developed for the detection of multiple AGAs, which the LODs for STR, KAN, NEO, and GEN were 4.74 ng/mL, 4.97 ng/mL, 2.99 ng/mL, and 4.42 ng/mL respectively. To improve the sensitivity of immunoassay, single-well carbon tubes (SWCNTs) were utilized as solid support for loading horseradish peroxidase-labelled goat anti-mouse antibody to obtain the multi-enzyme particles. After the optimization of usage of multi-enzyme particles and antibodies, the enhanced CLMIA was established and evaluated. The LODs were 1.25 ng/mL for STR, 0.64 ng/mL for KAN, 0.38 ng/mL for GEN, and 0.39 ng/mL for NEO, which was improved by threefold, sevenfold, 11-fold, and sevenfold compared with the conventional CLMIA developed. These methods presented higher specificity and repeatability. Finally, the enhanced CLMIA based on CNT-assisted multi-enzyme particles was utilized to analyze twenty-five milk samples from local market and dairy farm, which all the results were below the LOD. The enhanced CLMIA showed the great application potential for the detection of multiple targets simultaneously and provided efficient tool for the screening of pollutants in food.

Array-based microbial identification upon extracellular aminoglycoside residue sensing.

Sensitive and rapid identification of pathogenic microorganisms is of great importance for clinical diagnosis and treatment. In this study, we developed an ultrasensitive colorimetric sensor array (CSA) based on the interactions between aminoglycoside antibiotics (AMGs) and Ag nanoparticles decorated with ß-cyclodextrin (AgNPs@ß-CD) to discriminate microorganisms quickly and accurately. Microorganisms can absorb different amounts of AMGs after incubation. Upon the addition of AgNPs@ß-CD, the corresponding extracellular AMG residues will bind to AgNPs@ß-CD, leading to color changes due to the modifications in localized surface plasmon resonance. The array was developed using 4 AMGs as sensing elements and AgNPs@ß-CD as the colorimetric probe to generate a unique colorimetric response pattern for each microorganism. Standard chemometric methods indicated excellent discrimination among 20 microorganisms at low concentrations of 2 × 106 CFU/mL. Therefore, this ultrasensitive CSA can be used for microbial discrimination portably and efficiently. Importantly, the concentration of microbial discrimination by our array is much lower than that of prior CSAs. This method of extracellular residue sensing also provided a new strategy to improve the sensitivity of conventional CSA in the discrimination of microorganisms, to measure the amount of intercellular uptake of AMGs by microorganisms, and to screen drugs that can easily be accumulated by the pathogenic microorganisms.

Derivatization-free determination of aminoglycosides by CZE-UV in pharmaceutical formulations.

Aminoglycosides are a relevant class of antibiotics widely used by medics and veterinaries. There are a variety of reasons that make their determination relevant, such as quality control, environment and food contamination assessment, drug-release studies, among others. The lack of a chromophore makes aminoglycoside spectrophotometric detection particularly challenging, often requiring derivatization. In this work, an indirect detection method, making use of imidazole as a probe, applying CZE was successfully tested. It did not require derivatization, which simplified the sample preparation. Suitable figures of merit were obtained; recoveries between 95 and 105%, adequate repeatability and precision, correlation coefficients (r) above 0.998, and limits of detection (LODs) of 3.2 and 11 mg/L for gentamicin and paromomycin, respectively. As a proof-of-concept, it was also applied in a simple controlled release experiment that was well fitted using the Hill equation.

Low-cost and versatile analytical tool with purpose-made capillary electrophoresis coupled to contactless conductivity detection: Application to antibiotics quality control in Vietnam.

In this study, the development of our purpose-made capacitively coupled contactless conductivity detection (C4 D) for CE is reported. These systems have been employed as a simple, versatile, and cost-effective analytical tool. CE-C4 D devices, whose principle is based on the control of the ion movements under an electrical field, can be constructed even with a modest financial budget and limited infrastructure. A featured application was developed for quality control of antimicrobial drugs using CE-C4 D, with most recent work on determination of aminoglycoside and glycopeptide antibiotics being communicated. For aminoglycosides, the development of CE-C4 D methods was adapted to two categories. The first one includes drugs (liquid or powder form) for intravenous injection, containing either amikacin, streptomycin, kanamycin A, or kanamycin B. The second one covers drugs for eye drops (liquid or ointment form), containing either neomycin, tobramycin, or polymyxin. The CE-C4 D method development was also made for determination of some popular glycopeptide antibiotics in Vietnam, including vancomycin and teicoplanin. The best detection limit achieved using the developed CE-C4 D methods was 0.5 mg/L. Good agreement between results from CE-C4 D and the confirmation method (HPLC- Photometric Diode Array ) was achieved, with their result deviations less than 8% and 13% for aminoglycoside and glycopeptide antibiotics, respectively.

Simultaneous determination of ten aminoglycoside antibiotics in aquatic feeds by high-performance liquid chromatography quadrupole-orbitrap mass spectrometry with pass-through cleanup.

A simple and sensitive method has been established based on pass-through cleanup and high-performance liquid chromatography quadrupole-orbitrap mass spectrometry (HPLC-Q/Orbitrap MS) for the simultaneous determination of ten aminoglycosides (AGs) in aquatic feeds. The extraction solution and cleanup procedure had been optimized, and good sensitivity, accuracy, and precision were obtained. The calibration curves of AGs were linearity (R2 > 0.99) in the range of 2.0 to 200 µg/L (or 5.0 to 500 µg/L). The limits of detection of AGs were between 10 and 25 µg/kg. The recoveries of AGs ranged from 74.9% to 94.3%, and the intraday and interday relative standard deviations were less than 15%. Finally, this method was successfully applied to determine ten AGs in 30 aquatic feed samples. It might be the first time to use pass-through cleanup approach combined with HPLC-Q/Orbitrap MS method for AGs determination in aquatic feed samples.

Aptamer-based ellipsometric sensor for ultrasensitive determination of aminoglycoside group antibiotics from dairy products.

BACKGROUND: Residual antibiotics taken along with food consumed through the food chain are the main cause of the super-bacteria and may damage organs such as liver and kidney. Therefore, monitoring residual antibiotic levels of products in the food chain is both important and a requirement. Maximum residual limits for kanamycin and neomycin are 150 ng mL-1 and 500 ng mL-1 respectively, which are challenging for most sensor platforms. In this paper, a novel method is presented for the determination of antibiotics residues in animal-derived foods. RESULTS: Aptamer-based kanamycin and neomycin biosensors based on the spectroscopic ellipsometer and the surface plasmon resonance-enhanced total internal reflection ellipsometer methods as transducing element were developed. Detection limits of both sensor platforms were in the 0.1-1 nmol L-1 ranges, and the detection range was between the detection limit and 1000 nmol L-1 . CONCLUSION: Both ellipsometry-based aptasensors can be used as an alternative to the existing enzyme-linked immunosorbent assay-based method in terms of assay time (10 min), detection limit (0.22 ng mL-1 for neomycin and 0.048 ng mL-1 for kanamycin), and detection range. © 2020 Society of Chemical Industry.

Unexpected drug residuals in human milk in Ankara, capital of Turkey.

BACKGROUND: Breast milk is a natural and unique nutrient for optimum growth and development of the newborn. The aim of this study was to investigate the presence of unpredictable drug residues in mothers' milk and the relationship between drug residues and maternal-infant characteristics. METHODS: In a descriptive study, breastfed infants under 3 months of age and their mothers who applied for child health monitoring were enrolled for the study. Information forms were completed for maternal-infant characteristics, breastfeeding problems, crying and sleep characteristics of infants. Maternal and infant anthropometric measurements and maternal milk sample were taken. Edinburgh Postpartum Depression Scale was applied to mothers. RANDOX Infiniplex kit for milk was used for residual analysis. RESULTS: Overall, 90 volunteer mothers and their breastfed infants were taken into the study and the mean age of the mothers and their infants was 31.5 ± 4.2 years and 57.8 ± 18.1 days, respectively. Anti-inflammatory drug residues in breast milk were detected in 30.0% of mothers and all had tolfenamic acid. Overall, 94.4% had quinolone, 93.3% beta-lactam, 31.1% aminoglycoside and 13.3% polymycin residues. Drugs used during pregnancy or lactation period were not affected by the presence of residues. Edinburgh postpartum depression scores of mothers and crying and sleeping problems of infants were similar in cases with and without drug residues in breast milk. When controlling confounding factors, maternal body mass index alterations were detected to be significantly lower in mothers with anti-inflammatory drug residues in breast milk than in their counterparts (p = 0.017). CONCLUSIONS: Our study suggests that there are unpredictable drug residues in the milk of many mothers. Anti-inflammatory drug exposure might affect maternal weight change during the postpartum period. Further studies are required to evaluate the impact of drug residues on maternal and infant health.

A microbiological inhibition method for the rapid, broad-spectrum, and high-throughput screening of 34 antibiotic residues in milk.

In this study, we developed a microbiological inhibition method for the rapid screening of antibiotics in milk with Geobacillus stearothermophilus ATCC12980 as an indicator bacterium and an easy sample pretreatment. We observed that the limits of detection of the kit for 34 common antibiotic residues in milk, including ß-lactams (13), aminoglycosides (6), tetracyclines (4), sulfonamides (6), macrolides (4), lincosamides (1), were lower than or close to the maximum residue limits formulated by the European Union and China. Moreover, the false-positive rate was 1% and the false-negative rates were less than 5%. The ruggedness of the method (the reproducibility of detection capability of different batches of medium) met requirements at determined levels and residual limits. The shelf life of the kit was more than 6 mo at 4°C. Additionally, we observed good correlations between the kit results and ultra-high-performance liquid chromatography-tandem mass spectrometry results for incurred milk (samples taken from animals treated with antibiotics according to the pre-slaughter medication data), which indicated that the kit was reliable for screening antibiotics in incurred samples. In conclusion, the kit has a broad application potential with high sensitivity, specificity, and reproducibility, stability, and reliability, combined with simple operation, low cost, and high-throughput capacity.

Simultaneous analysis of aminoglycosides with many other classes of drug residues in bovine tissues by ultrahigh-performance liquid chromatography-tandem mass spectrometry using an ion-pairing reagent added to final extracts.

The way to maximize scope of analysis, sample throughput, and laboratory efficiency in the monitoring of veterinary drug residues in food animals is to determine as many analytes as possible as fast as possible in as few methods as possible. Capital and overhead expenses are also reduced by using fewer instruments in the overall monitoring scheme. Traditionally, the highly polar aminoglycoside antibiotics require different chromatographic conditions from other classes of drugs, but in this work, we demonstrate that an ion-pairing reagent (sodium 1-heptanesulfonate) added to the combined final extracts from two sample preparation methods attains good separation of 174 targeted drugs, including 9 aminoglycosides, in the same 10.5-min ultrahigh-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) analysis. The full method was validated in bovine kidney, liver, and muscle tissues according to US regulatory protocols, and 137-146 (79-84%) of the drugs gave between 70 and 120% average recoveries with ≤ 25% RSDs in the different types of tissues spiked at 0.5, 1, and 2 times the regulatory levels of interest (10-1000 ng/g depending on the drug). This method increases sample throughput and the possible number of drugs monitored in the US National Residue Program, and requires only one UHPLC-MS/MS method and instrument for analysis rather than two by the previous scheme. Graphical abstract Outline of the streamlined approach to monitor 174 veterinary drugs, including aminoglycosides, in bovine tissues by combining two extracts of the same sample with an ion-pairing reagent for analysis by UHPLC-MS/MS.

Simultaneous screening and quantification of aminoglycoside antibiotics in honey using mixed-mode liquid chromatography with quadrupole time-of-flight mass spectroscopy with heated electrospray ionization.

An analytical method based on liquid chromatography with quadrupole time-of-flight mass spectrometry has been developed for the simultaneous determination of six aminoglycoside antibiotics in honey. The sample pretreatment included extraction with aqueous trichloroacetic acid followed by solid-phase extraction on Strata-X polymeric reversed phase cartridges. Liquid chromatography separation was performed on an Obelisc R zwitterionic type mixed-mode column. An ionBooster™ heated electrospray source was used and showed enhanced ionization efficiency in comparison to a conventional electrospray source. The observed signal enhancement ranged from 3- (neomycin) to 16-fold (gentamicin C1). A data-dependent mass spectrometry acquisition approach was employed, in which the full mass spectrometry dataset provided quantification and a scheduled precursor list was used to trigger an alternating data-dependent acquisition of MS2 spectra for confirmation purposes. The described method was validated in accordance to CD 2002/657/EC. Decision limit values were in the range 11.2-33.6 ng/g, and satisfactory performance characteristics were obtained for recovery (65-76%), repeatability (3.8-7.3%), and linearity (≥0.995). The method was applied to the analysis of 49 real honey samples from the country of Georgia. Streptomycin was detected in two samples at 117 and 35 ng/g, and gentamicin C1 was detected in one sample at 32 ng/g.

Susceptibility of Malassezia pachydermatis to aminoglycosides.

Previous studies have evaluated the action of gentamicin against Malassezia pachydermatis. The aim of this study was to evaluate in vitro susceptibility of M. pachydermatis to the aminoglycosides- gentamicin, tobramycin, netilmicin and framycetin. The minimum inhibitory concentration (MIC) of gentamicin was determined following methods M27-A3 microdilution and Etest® . The Etest® was used to determine the minimum inhibitory concentration (MIC) of the tobramycin and netilmicin. The Kirby-Bauer test was used to determine the antibiotic susceptibility to the framycetin. The MIC50 and MIC90 were 8.12 µg/mL and 32.5 µg/mL by microdilution method for gentamicin. The MIC50, determined by the Etest® , was 8 µg/mL for gentamicin and netilmicin and 64 µg/mL for tobramycin. The MIC90 was 16 and 32 µg/mL for gentamicin and netilmicin respectively. The MIC90 was outside of the detectable limits for tobramycin. To framycetin, 28 strains (40%) of the 70 M. pachydermatis isolates tested showed a diameter of 22 mm, 22 strains (31.42%) showed a diameter of 20 mm, 16 strains showed a diameter of ≤ 18 mm, and only 5.71% of the isolates showed a diameter of ≥ 22 mm. This study provides evidence of high in vitro activity of the aminoglycosides-gentamicin, tobramycin, netilmicin and framycetin against M. pachydermatis. For gentamicin Etest® showed similar values of MIC50 y MIC90 that the obtained by microdilution method. We considered Etest® method could be a good method for these calculations with aminoglycosides.

High throughput LSPR and SERS analysis of aminoglycoside antibiotics.

Aminoglycoside antibiotics are used in the treatment of infections caused by Gram-negative bacteria, and are often dispensed only in severe cases due to their adverse side effects. Patients undergoing treatment with these antibiotics are therefore commonly subjected to therapeutic drug monitoring (TDM) to ensure a safe and effective personalised dosage. The ability to detect these antibiotics in a rapid and sensitive manner in human fluids is therefore of the utmost importance in order to provide effective monitoring of these drugs, which could potentially allow for a more widespread use of this class of antibiotics. Herein, we report on the detection of various aminoglycosides, by exploiting their ability to aggregate gold nanoparticles. The number and position of the amino groups of aminoglycoside antibiotics controlled the aggregation process. We investigated the complementary techniques of surface enhanced Raman spectroscopy (SERS) and localized surface plasmon resonance (LSPR) for dual detection of these aminoglycoside antibiotics and performed an in-depth study of the feasibility of carrying out TDM of tobramycin using a platform amenable to high throughput analysis. Herein, we also demonstrate dual detection of tobramycin using both LSPR and SERS in a single platform and within the clinically relevant concentration range needed for TDM of this particular aminoglycoside. Additionally we provide evidence that tobramycin can be detected in spiked human serum using only functionalised nanoparticles and SERS analysis.

Characterization of fortimicin aminoglycoside profiles produced from Micromonospora olivasterospora DSM 43868 by high-performance liquid chromatography-electrospray ionization-ion trap-mass spectrometry.

In this study, an efficient high-performance liquid chromatography (HPLC)-electrospray ionization (ESI)-ion trap-tandem mass spectrometry (MS/MS) was developed for the identification of the biosynthetic congeners involved in the aminocyclitol aminoglycosidic fortimicin pathway from Micromonospora olivasterospora fermentation. The usage of both acid extraction (pH ∼2.5) followed by an cationic-exchanging SPE cleanup and pentafluoropropionic acid mediated ion-pairing chromatography with ESI-ion trap-MS/MS detection was determined to be sufficiently practical to profile the fortimicin (FOR) congeners produced in a culture broth. The limit of the quantification for the fortimicin A (FOR-A) standard spiked in the culture broth was ∼1.6 ng mL(-1). The average recovery rate was 93.6%, and the intra- and inter-day precisions were <5% with accuracy in the range from 87.1 to 94.2%. Moreover, the epimeric mixtures including FOR-KH, FOR-KR, and FOR-B were separately resolved through a macrocyclic glycopeptide (teicoplanin)-bonded chiral column. As a result, ten natural FOR pseudodisaccharide analogs were identified and semi-quantified in descending order as follows: FOR-A, FOR-B, DCM, FOR-KH plus FOR-KR, FOR-KK1, FOR-AP, FOR-KL1, FOR-AO, and FOR-FU-10. This is the first report on both the simultaneous characterization of diverse structurally closely related FORs derived from bacterial fermentation using HPLC-ESI-ion trap-MS/MS analysis and the chromatographic separation of the three FOR epimers.

Mixed-mode liquid chromatography coupled to tandem mass spectrometry for the analysis of aminoglycosides in meat.

A novel LC-MS/MS method has been developed for the determination of 13 aminoglycoside antibiotics in meat products. Among the chromatographic columns tested, the mixed-mode Obelisc R provided the best performance. Electrospray has been used for the coupling of the LC and the effect of temperature on the ionization has been evaluated. The mass spectra of AGs have been studied in order to select the most adequate precursor and product ions for quantitation and confirmation in SRM mode, showing that the single charged [M+H](+) provided better precisions than the double charged [M+2H](2+). Accurate mass measurements have been performed in order to confirm the molecular composition of the product ions, allowing the establishment of a new mechanism for some product ions of STR and DHSTR. A sample treatment based on an extraction and a SPE clean-up has been applied to a wide variety of meat products such as frankfurters; sausages; and minced meat of pork, veal, and chicken. Method limits of quantitation in the low microgram per kilogram level (1-50 µg kg(-1)), precisions %RSD below 15 % and accuracies expressed as relative errors below 23 % have been obtained, making the proposed method suitable for routine analysis.

A rapid SPE-based analytical method for UPLC/MS/MS determination of aminoglycoside antibiotic residues in bovine milk, muscle, and kidney.

An SPE-based cleanup protocol was developed for ultra-performance LC (UPLC)/MS/MS determination of residues of the common aminoglycoside antibiotics streptomycin, dihydrostreptomycin, neomycin, and gentamicin in bovine milk, kidney, and muscle. Recoveries for all compounds except neomycin ranged from 80 to 104% for all matrixes studied; recoveries for neomycin ranged from 71 to 84%. Intraday and interday precision data were under 15% for all sample matrixes. Compared with other recently reported cleanup methods, less sample is required, the use of potentially dangerous reagents is minimized, and fewer manipulations are required by the analyst. A high throughput 96-well plate format was used for SPE cleanup and UPLC/MS analysis.

DNA-aptamers binding aminoglycoside antibiotics.

Aptamers are short, single stranded DNA or RNA oligonucleotides that are able to bind specifically and with high affinity to their non-nucleic acid target molecules. This binding reaction enables their application as biorecognition elements in biosensors and assays. As antibiotic residues pose a problem contributing to the emergence of antibiotic-resistant pathogens and thereby reducing the effectiveness of the drug to fight human infections, we selected aptamers targeted against the aminoglycoside antibiotic kanamycin A with the aim of constructing a robust and functional assay that can be used for water analysis. With this work we show that aptamers that were derived from a Capture-SELEX procedure targeting against kanamycin A also display binding to related aminoglycoside antibiotics. The binding patterns differ among all tested aptamers so that there are highly substance specific aptamers and more group specific aptamers binding to a different variety of aminoglycoside antibiotics. Also the region of the aminoglycoside antibiotics responsible for aptamer binding can be estimated. Affinities of the different aptamers for their target substance, kanamycin A, are measured with different approaches and are in the micromolar range. Finally, the proof of principle of an assay for detection of kanamycin A in a real water sample is given.

Development of a Fourier transform infrared spectroscopy coupled to UV-Visible analysis technique for aminosides and glycopeptides quantitation in antibiotic locks.

Antibiotic Lock technique maintains catheters' sterility in high-risk patients with long-term parenteral nutrition. In our institution, vancomycin, teicoplanin, amikacin and gentamicin locks are prepared in the pharmaceutical department. In order to insure patient safety and to comply to regulatory requirements, antibiotic locks are submitted to qualitative and quantitative assays prior to their release. The aim of this study was to develop an alternative quantitation technique for each of these 4 antibiotics, using a Fourier transform infrared (FTIR) coupled to UV-Visible spectroscopy and to compare results to HPLC or Immunochemistry assays. Prevalidation studies permitted to assess spectroscopic conditions used for antibiotic locks quantitation: FTIR/UV combinations were used for amikacin (1091-1115cm(-1) and 208-224nm), vancomycin (1222-1240cm(-1) and 276-280nm), and teicoplanin (1226-1230cm(-1) and 278-282nm). Gentamicin was quantified with FTIR only (1045-1169cm(-1) and 2715-2850cm(-1)) due to interferences in UV domain of parabens, preservatives present in the commercial brand used to prepare locks. For all AL, the method was linear (R(2)=0.996 to 0.999), accurate, repeatable (intraday RSD%: from 2.9 to 7.1% and inter-days RSD%: 2.9 to 5.1%) and precise. Compared to the reference methods, the FTIR/UV method appeared tightly correlated (Pearson factor: 97.4 to 99.9%) and did not show significant difference in recovery determinations. We developed a new simple reliable analysis technique for antibiotics quantitation in locks using an original association of FTIR and UV analysis, allowing a short time analysis to identify and quantify the studied antibiotics.

[Inducible specific lux-biosensors for the detection of antibiotics: construction and main parameters].

Based on Escherichia coli, highly sensitive specific lux-biosensors for the detection of tetracycline and beta-lactam antibiotics, quinolones, and aminoglycosides have been obtained. To make biosensors, bacteria were used that contained fungal plasmids pTetA'::lux, pAmpC'::lux, pColD'::lux, and plbpA'::lux, in which transcription of the reporter Photorhabdus luminescens luxCDABE genes occurred from the inducible promoters of the tetA, ampC, cda, and ibpA genes, respectively. The main parameters (threshold sensitivity and response time) of lux-biosensors were measured. The high specificity of biosensors responding only to antibiotics of a certain type was demonstrated.

High-pH mobile phase in reversed-phase liquid chromatography-tandem mass spectrometry to improve the separation efficiency of aminoglycoside isomers.

In chromatography, the use of extreme conditions can often lead to unique separation selectivity. In this study, a highly basic mobile phase (pH > 11), which is not typically employed for reversed-phase liquid chromatography (RPLC), was utilized in RPLC-tandem mass spectrometry (MS/MS) to achieve effective separation between electrically neutral bases of aminoglycosides (AGs). A mixture of AGs was simultaneously analyzed using 500 mmol L-1 ammonia aqueous solution (pH 11.8) as the mobile phase. A total of 11 AGs, including 2 stereoisomers of neomycin (B and C) and 5 structurally similar components of gentamicin (C1, C1a, C2, C2a, and C2b), were completely separated for the first time. The high separation performance for AGs was mainly due to two factors: First, slight differences in hydrophobicity among the AGs were significantly enhanced at a high pH by the complete acid dissociation of amines. Second, the high pH of the mobile phase minimized any electrostatic interactions between the AGs and residual silanol groups in the stationary phase, resulting in extremely sharp peaks for the AGs. The sensitivity of spectinomycin decreased by more than 20% when using the highly basic mobile phase (pH 11.8) due to its degradation, therefore, a mixture of 10 AGs was analyzed with 250 mmol L-1 ammonia aqueous solution (pH 11.5) with less degradation as the optimum condition. The developed analytical method could be used to determine the concentrations of trace AGs in milk with high accuracy and precision. Thus, RPLC-MS/MS using a high-pH mobile phase has great potential for the efficient separation of basic compounds containing amino sugars such as AGs.