Source Tracing of Kidney Injury via the Multispectral Fingerprint Identified by Machine Learning-Driven Surface-Enhanced Raman Spectroscopic Analysis.

Early diagnosis of drug-induced kidney injury (DIKI) is essential for clinical treatment and intervention. However, developing a reliable method to trace kidney injury origins through retrospective studies remains a challenge. In this study, we designed ordered fried-bun-shaped Au nanocone arrays (FBS NCAs) to create microarray chips as a surface-enhanced Raman scattering (SERS) analysis platform. Subsequently, the principal component analysis (PCA)-two-layer nearest neighbor (TLNN) model was constructed to identify and analyze the SERS spectra of exosomes from renal injury induced by cisplatin and gentamycin. The established PCA-TLNN model successfully differentiated the SERS spectra of exosomes from renal injury at different stages and causes, capturing the most significant spectral features for distinguishing these variations. For the SERS spectra of exosomes from renal injury at different induction times, the accuracy of PCA-TLNN reached 97.8% (cisplatin) and 93.3% (gentamicin). For the SERS spectra of exosomes from renal injury caused by different agents, the accuracy of PCA-TLNN reached 100% (7 days) and 96.7% (14 days). This study demonstrates that the combination of label-free exosome SERS and machine learning could serve as an innovative strategy for medical diagnosis and therapeutic intervention.

Synthesis of Gentamicins C1, C2, and C2a and Antiribosomal and Antibacterial Activity of Gentamicins B1, C1, C1a, C2, C2a, C2b, and X2.

Complementing our earlier syntheses of the gentamicins B1, C1a, C2b, and X2, we describe the synthesis of gentamicins C1, C2, and C2a characterized by methyl substitution at the 6'-position, and so present an alternative access to previous chromatographic methods for accessing these sought-after compounds. We describe the antiribosomal activity of our full set of synthetic gentamicin congeners against bacterial ribosomes and hybrid ribosomes carrying the decoding A site of the human mitochondrial, A1555G mutant mitochondrial, and cytoplasmic ribosomes and establish structure-activity relationships with the substitution pattern around ring I to antiribosomal activity, antibacterial resistance due to the presence of aminoglycoside acetyl transferases acting on the 6'-position in ring I, and literature cochlear toxicity data.

Novel CdS/MOF Cathodic Photoelectrochemical (PEC) Platform for the Detection of Doxorubicin Hydrochloride and Gentamicin Sulfate.

Nanomaterial selection is critical for photoelectrochemical (PEC) sensing. In this report, a novel cathodic photoelectrochemical (PEC) strategy was proposed for the detection of doxorubicin hydrochloride (Dox) and gentamicin sulfate (CN). The photocathode was synthesized by noncovalently coupling cadmium sulfide (CdS) to the porphyrin-derived metal-organic framework (CdS@PCN-224). This type of assembly created a pleasant interface for the combination of doxorubicin hydrochloride and gentamicin sulfate, resulting in a good CdS@PCN-224 donor-acceptor system. When compared to a single optoelectronic material, its photocurrent is enhanced by unprecedented nine times. This research could pave the way for the realization of PCN-224's enormous potential in PEC sensing.

Analysis of histamine and sisomicin in gentamicin: Search for the causative agents of adverse effects.

In 1998, the aminoglycoside antibiotic gentamicin sulfate caused several cases of deaths in the United States, after the switch from twice- to once-daily application. Endotoxins were discussed as the cause for the adverse effects and sisomicin was identified as the lead impurity; batches containing sisomicin were contaminated with more impurities and were responsible for the fatalities. In 2016, anaphylactic reactions in horses, and later in humans with one fatality, were observed after application of gentamicin sulfate contaminated with histamine. To determine whether histamine was responsible for the 1990s death cases as well, histamine was quantified by means of liquid chromatography-tandem mass spectrometry (LC-MS/MS) in 30 samples of gentamicin sulfate analyzed in previous studies. Furthermore, a relative quantification of sisomicin was performed to check for a correlation between histamine and the lead impurity. A maximum amount of 11.52 ppm histamine was detected, which is below the limit for anaphylactic reactions of 16 ppm, and no correlation of the two impurities was observed. However, the European Medicines Agency recommends a stricter limit with regard to the maximum single dose of gentamicin sulfate to reach a greater gap between the maximum histamine exposition of 4.3 µg and the quantity known to cause hypotension of 7 µg. The low amounts of histamine and the fact that there is no connection with the contamination with sisomicin showed that histamine was not the cause for the death cases in the United States in 1998, and endotoxins remain the most probable explanation.

A Paper-Based Colorimetric Aptasensor for the Detection of Gentamicin.

Antibiotics are classes of antimicrobial substances that are administered widely in the field of veterinary science to promote animal health and feed efficiency. Cattle-administered antibiotics hold a risk of passing active residues to milk, during the milking process. This becomes a public health concern as these residues can cause severe allergic reactions to sensitive groups and considerable economic losses to the farmer. Hence, to ensure that the produced milk is safe to consume and adheres to permissible limits, an on-farm quick and reliable test is essential. This study illustrates the design and development of a microfluidic paper biosensor as a proof-of-concept detection system for gentamicin in milk. Localized surface plasmon resonance (LSPR) properties of gold nanoparticles have been explored to provide the user a visual feedback on the test, which was also corroborated by RGB analysis performed using Image J. The assay involves the use of a short stretch of single stranded DNA, called aptamer, which is very specific to the gentamicin present in the milk sample. The camera-based LOD for the fabricated paper device for milk samples spiked with gentamicin was calculated to be 300 nM, with a reaction time of 2 min.

Na+-NQR Confers Aminoglycoside Resistance via the Regulation of l-Alanine Metabolism.

Sodium-translocating NADH:quinone oxidoreductase (Na+-NQR) functions as a unique redox-driven sodium pump, generating membrane potential, which is related to aminoglycoside antibiotic resistance. However, whether it modulates other metabolisms to confer antibiotic resistance is unknown. The present study showed that loss of nqrA or nqrF led to differential metabolomes with elevated resistance to aminoglycoside antibiotics. Decreased alanine, aspartate, and glutamate metabolism and depressed abundance of alanine were characterized as the most impacted pathway and crucial biomarker, respectively. Further data showed that higher viability was detected in ΔnqrA and ΔnqrF mutant strains than their parent strain ATCC 33787 in the presence of gentamicin but recovered by exogenous l-alanine. It proceeds by the following events. The loss of nqrA or nqrF led to the decrease of membrane potential, ATPase activity, and then ATP and cyclic AMP (cAMP), which reduced the cAMP/CRP (cAMP receptor protein) complex. The reduced cAMP/CRP complex promoted l-alanine catabolism and inhibited l-alanine anabolism, causing reduced levels of alanine. Reduced alanine affected the expression of antiporter families Atp and Mnh genes. Our results suggest a novel mechanism by which the Na+-NQR system regulates antibiotic resistance via l-alanine metabolism in a cAMP/CRP complex-dependent manner.IMPORTANCE The Na+-NQR complex functions as a unique redox-driven sodium pump, generating membrane potential directly. However, whether it mediates generation of membrane potential indirectly is unknown. The present study shows that the Na+-NQR complex impacts membrane potential through other antiporter families Atp and Mnh. It proceeds by ATP and then cAMP/CRP regulon, which inhibits l-alanine catabolism and promotes l-alanine anabolism. When the Na+-NQR complex is reduced as in antibiotic-resistant bacteria, l-alanine is depressed, which is related to the antibiotic resistance phenotypes. However, exogenous l-alanine reverts the phenotype and promotes antibiotic-mediated killing. These findings suggest a novel mechanism by which the Na+-NQR system regulates antibiotic resistance via l-alanine metabolism in a cAMP/CRP complex-dependent manner.

Analytical validation of a quantitative method for therapeutic drug monitoring on the Alinity®c Abbott.

OBJECTIVE: The aim of this study was to evaluate the analytical performance of the Alinity®c Abbott compared to the Architect® immunoassay system for the determination of drugs having a narrow therapeutic index. METHODS: Valproic acid, amikacin, gentamicin, phenobarbital and vancomycin were analyzed using Particle-Enhanced Turbidimetric Inhibitor Immunoassay (Petinia), phenytoin and theophylline were analyzed using an immunoenzymatic method and a colorimetric method was performed to quantify lithium. The methods were validated according to the total error approach. Seven validation standards were analyzed in quintuplet during four days to establish the limits of the methods. Dilution integrity and interferences (hemolysis and high concentrations of bilirubin and lipids) were also tested. Depending on the analyte, the results obtained for twenty to forty patients on the Alinity® were compared to those obtained on the Architect®. RESULTS: The bias and the coefficients of variation for repeatability and for intermediate precision were lower than 15% for all drugs. Accuracy profiles were acceptable (acceptance limits fixed at 30%) in the validated ranges. The lower limits of quantification (LLOQ) were similar to those determined by Abbott except for gentamicin for which we determined a LLOQ at 1.22 mg/L while Abbott determined it at 0.5 mg/L. All assays diluted linear and analyte concentrations were not affected by interferences. Concentrations obtained for real samples on the Alinity®c are comparable to those obtained on the Architect®ci. CONCLUSIONS: The analytical validation of a method suitable for therapeutic drug monitoring of drugs on the Alinity®c meets the requirements of European Medicines Agency.

Coupling of liquid-liquid extraction and mathematical filtration techniques for the separation and quantification of five components in semisolid dosage form with severely overlapped spectra.

Quadriderm cream was a combination of four components; Clioquinol (CLIO), Betamethasone (BETA), Tolnaftate (TOL), Gentamicin (GEN) in addition to the preservative Chlorocresol (CC). Four components CLIO, TOL, BETA, and CC were extracted in methanol and determined by mathematic filtration spectrophotometric techniques. The partially overlapped spectrum of CLIO was determined by constant value, constant multiplication, and concentration value methods then eliminated via spectrum subtraction (SS) to get the resolved ternary mixture of TOL, BETA, and CC with severely overlapping spectra. TOL was determined by derivative ratio at zero crossing point of BETA using CC as a divisor. While, BETA could be determined using TOL as a divisor at zero crossing of CC. BETA and CC were obtained using novel (DD1FS) followed by SS. By applying these novel procedures, the DD1 spectrum of each component alone was recovered where Pmax-min was directly proportional to its concentration. Liquid-liquid extraction technique was used for the semisolid dosage form where GEN was extracted with a mixture of chloroform: water (50:50, v/v); and the induced fluorescence obtained by derivatization with o-phthalaldehyde was measured at 419 nm after excitation at 359 nm. Accuracy and precision testing of the developed methods showed good results. Specificity of the methods was ensured and was successfully applied for the analysis of pharmaceutical formulation of the five components in combination. ICH guidelines were used for validation of the proposed methods. Statistical data were calculated, and the results were satisfactory revealing no significant difference regarding accuracy and precision.

Green synthesis of hesperidin coated silver nanoparticles for colorimetric detection of gentamicin sulfate in real samples.

Gentamicin sulfate (GEN), a well-known broad-spectrum antibiotic is mostly administered through intramuscular injections and entirely excreted in un-metabolized form through urination from patient's body. Quantitative detection of GEN by direct UV absorption is usually challenging due to lack of chromophores and fluorophores in structure. The current study described the hesperidin coated silver nanoparticles (HSPAgNPs) based novel colorimetric quantitative assay for GEN. HSPAgNPs, based colorimetric detection involved a transition from characteristic yellow colour to blackish brown upon addition of GEN, accompanied by a significant quenching in localized surface plasmon resonance (LSPR) band at λmax 398 nm. Moreover, the synthesized HSPAgNPs were employed to rapid and quantitative detection of GEN in concentration range of 5 to 100 µM. Limit of detection (LOD) and limit of quantification (LOQ) was calculated by standard deviation of the ordinate intercept and slope of the regression line and estimated to be 6.89 µM and 20.88 µM respectively, with a linear correlation factor R2 equal to 0.9990 which strictly followed Beer's law. Furthermore, the utility and effectiveness of HSPAgNPs was also explored for selective recognition of GEN in tap water, serum, human blood plasma and urine.

Sequential sandwich immunoassay for simultaneous detection in trace samples using single-channel surface plasmon resonance.

To analyze multiple analytes in trace samples, low-dosage and high efficiency are crucial in many common cases. Herein, we developed a facile method using a single-channel surface plasmon resonance (SPR)-based biosensor for the simultaneous detection of gentamicin (GEN) and melamine (MEL) in milk and serum with only one sample injection. Based on a sandwich immunoassay, non-interfering antibodies against GEN from mouse (AbGEN) and against MEL from rabbit (AbMEL) were chosen to capture the analytes. Secondary antibodies against mouse (AbM) and rabbit (AbR) were used to bind with AbGEN and AbMEL to determine the concentrations of GEN and MEL on a single channel of an SPR sensor. All of the detection process could be done in 10 min with 50 µL of sample injection. According to the response shifts of AbM and AbR, two standard curves for GEN and MEL were obtained successively, with the limit of detection (LOD) values at 4.4 ng mL-1 and 1.3 ng mL-1, respectively. Moreover, the feasibility was determined by spiking milk and serum samples with GEN and MEL, with recoveries in the range of 81.6%-118.0%. Importantly, the analytes can be substituted by others for much more applications. This method is also expected to multiply the detection efficiency of multi-channel SPR biosensors with low-dosage samples in the future.

An integrated microfluidic system for antimicrobial susceptibility testing with antibiotic combination.

Polypharmacy is routinely administered to fight severe infections, though it has led to rampant multi-drug resistance in many bacterial strains. Preferably, antimicrobial susceptibility testing (AST) would be carried out prior to antibiotic prescription, though it is generally thought to be too complex and labor-intensive. In order to assist clinicians with better antibiotic administration for the effective treatment of bacterial infections, an integrated microfluidic system (IMS) capable of automating AST for 1-2 antibiotics against clinical bacterial pathogens was developed herein. Accurate determination of the minimum and fractional inhibitory concentrations of vancomycin, gentamicin, and linezolid were determined by assaying growth of two clinical methicillin-resistant Staphylococcus aureus isolates via a colorimetric assay on-chip. By applying various antibiotic combinations against a single pathogen in multiple chambers, the IMS could identify the optimal drug combination and the minimum effective dosage by evaluating the fractional inhibitory concentration index. This IMS possessed several advantages over conventional methods, including (1) a 50% reduction in bacterial sample and reagent volume (<50 µL per well), (2) less potential for human error due to its automatic nature, (3) faster liquid manipulation time by integrating the microfluidic components rather than labor-intensive process, and (4) straightforward result interpretation via colorimetric change instead of turbidity degree. Personalized medicine for treatment of bacterial infections may therefore be realized using this IMS.

Engineered magnetosomes fused to functional molecule (protein A) provide a highly effective alternative to commercial immunomagnetic beads.

BACKGROUND: Magnetosomes (also called bacterial magnetic nanoparticles; BMPs) are biomembrane-coated nanoparticles synthesized by magnetotactic bacteria (MTB). Engineered BMPs fused to protein A (termed ∆F-BMP-FA) bind antibodies (Abs) automatically, and thus provide a series of potential advantages. However, no report so far has systematically evaluated functional applicability of genetically engineered BMPs. RESULTS: We evaluated properties of ∆F-BMP-FA, and developed/optimized culture methods for host strain Magnetospirillum gryphiswaldense ΔF-FA, ∆F-BMP-FA extraction conditions, conditions for Ab conjugation to ∆F-BMP-FA surface, and procedures for antigen detection using ∆F-BMP-FA/Ab complexes (termed BMP-A-Ab). Fed-batch culture for 36 h in a 42-L fermentor resulted in yields (dry weight) of 2.26 g/L for strain ΔF-FA and 62 mg/L for ∆F-BMP-FA. Optimal wash cycle number for ∆F-BMP-FA purification was seven, with magnetic separation following each ultrasonication step. Fusion of protein A to BMPs resulted in ordered arrangement of Abs on BMP surface. Linkage rate 962 µg Ab per mg ∆F-BMP-FA was achieved. BMP-A-Ab were tested for detection of pathogen (Vibrio parahaemolyticus; Vp) surface antigen and hapten (gentamicin sulfate). Maximal Vp capture rate for BMP-A-Ab was 90% (higher than rate for commercial immunomagnetic beads), and detection sensitivity was 5 CFU/mL. ∆F-BMP-FA also bound Abs from crude mouse ascites to form complex. Lowest gentamicin sulfate detection line for BMP-A-Ab was 0.01 ng/mL, 400-fold lower than that for double Ab sandwich ELISA, and gentamicin sulfate recovery rate for BMP-A-Ab was 93.2%. CONCLUSION: Our findings indicate that engineered BMPs such as ∆F-BMP-FA are inexpensive, eco-friendly alternatives to commercial immunomagnetic beads for detection or diagnostic immunoassays, and have high Ab-conjugation and antigen-adsorption capacity.

Insufficient harmonization of antibiotics assays - Polish experience with an external quality assessment program in the years 2011-2018.

INTRODUCTION: Treatment with vancomycin and gentamycin requires strict monitoring of its serum concentration for proper dosage optimization. This study aimed to assess the quality and the harmonization of antibiotics assays in Polish laboratories. MATERIALS AND METHODS: 413 results of vancomycin and 148 results of gentamycin assays obtained from Polish laboratories in 30 international external quality assessment (EQA) surveys carried out from March 2011 to May 2018 were analyzed. RESULTS: Interlaboratory robust coefficients of variation (rCVs) in particular surveys comprised between 1.3 and 47.2% for vancomycin, and between 1.8 and 34.2% for gentamycin. The percentage of the results with the difference above acceptable limit ±10% from the target value established for own method group was 25.7% for vancomycin and 25.6% for gentamycin. When the difference was established according to target value for all methods, the percentage of results outside the acceptable limit was 2-fold higher on average (54.8% for vancomycin and 43.2% for gentamycin). The comparison of target values for methods revealed statistically significant differences between analytical systems used (p < .0001). The highest difference was 40% for vancomycin and 12% for gentamycin. CONCLUSIONS: The present analysis revealed high dispersion of the antibiotics assays results in Polish laboratories. Moreover, vancomycin and gentamycin results differed significantly in a way dependent on the analytical system used. There appear to be an urgent need for harmonization of methods used for vancomycin and gentamycin measurement.

Alternative Enzyme Protection Assay To Overcome the Drawbacks of the Gentamicin Protection Assay for Measuring Entry and Intracellular Survival of Staphylococci.

Precise enumeration of living intracellular bacteria is the key step to estimate the invasion potential of pathogens and host immune responses to understand the mechanism and kinetics of bacterial pathogenesis. Therefore, quantitative assessment of host-pathogen interactions is essential for development of novel antibacterial therapeutics for infectious disease. The gentamicin protection assay (GPA) is the most widely used method for these estimations by counting the CFU of intracellular living pathogens. Here, we assess the longstanding drawbacks of the GPA by employing an antistaphylococcal endopeptidase as a bactericidal agent to kill extracellular Staphylococcus aureus We found that the difference between the two methods for the recovery of intracellular CFU of S. aureus was about 5 times. We prove that the accurate number of intracellular CFU could not be precisely determined by the GPA due to the internalization of gentamicin into host cells during extracellular bacterial killing. We further demonstrate that lysostaphin-mediated extracellular bacterial clearance has advantages for measuring the kinetics of bacterial internalization on a minute time scale due to the fast and tunable activity and the inability of protein to permeate the host cell membrane. From these results, we propose that accurate quantification of intracellular bacteria and measurement of internalization kinetics can be achieved by employing enzyme-mediated killing of extracellular bacteria (enzyme protection assay [EPA]) rather than the host-permeative drug gentamicin, which is known to alter host physiology.

A Q-TOF LC/MS method for identification and quantitation of Histamine in the antibiotic Gentamicin at ppm level: Validation and uncertainty evaluation.

Adverse reactions have been reported for antibiotics produced via fermentation with fish peptone due to Histamine contamination. Just few micrograms of Histamine can result in adverse reactions when administered intravenously. Thus in this paper a new method for identification and quantitation of Histamine at ppm levels in the antibiotic Gentamicin is described. The method is based on separation of Histamine from Gentamicin and other excipients present in the drug matrix, by hydrophilic interaction liquid chromatography (HILIC) coupled to a Q-TOF/MS detector; quantitation is based on the standard addition approach. The method was validated for repeatability, inter-day precision, specificity, accuracy (relative and absolute bias) linearity, limit of detection and quantitation. Uncertainty was estimated and evaluated by comparison with values expected according to the Horwitz theory. The method showed satisfactory performances and good sensitivity, reaching a limit of quantitation of approximately 1 ppm. The method proposed can be a starting point for the development of Histamine quantitation methods in other antibiotics or even in other medicinal products which active ingredient is produced via fermentation in presence of fish peptone.

Comparison of Elution Characteristics and Compressive Strength of Biantibiotic-Loaded PMMA Bone Cement for Spacers: Copal® Spacem with Gentamicin and Vancomycin versus Palacos® R+G with Vancomycin.

PURPOSE: Copal® spacem is a new PMMA bone cement for fabricating spacers. This study compares elution of gentamicin, elution of vancomycin, and compressive strength of Copal® spacem and of Palacos® R+G at different vancomycin loadings in the powder of the cements. We hypothesized that antibiotic elution of Copal® spacem is superior at comparable compressive strength. METHODS: Compression test specimens were fabricated using Copal® spacem manually loaded with 0.5 g gentamicin and additionally 2 g, 4 g, and 6 g of vancomycin per 40 g of cement powder (COP specimens) and using 0.5 g gentamicin premixed Palacos® R+G manually loaded with 2 g, 4 g, and 6 g of vancomycin per 40 g of cement powder (PAL specimens). These specimens were used for determination of gentamicin and vancomycin elution (in fetal calf serum, at 22°C) and for determination of compressive strength both prior and following the elution tests. RESULTS: Cumulative gentamicin concentrations (p < 0.005) and gentamicin concentration after 28 days (p ≤ 0.043) were significantly lower for COP specimens compared to PAL specimens. Cumulative vancomycin concentrations were significantly higher (p ≤ 0.043) for COP specimens after the second day. Vancomycin concentrations after 28 days were not significantly higher for the Copal specimens loaded with 2 g and 4 g of vancomycin. Compressive strength was not significantly different between COP specimens and PAL specimens before elution tests. Compressive strength after the elution tests was significantly lower (p = 0.005) for COP specimens loaded with 2 g of vancomycin. CONCLUSION: We could not demonstrate consistent superior antibiotic elution from Copal® spacem compared to Palacos® R+G for fabricating gentamicin and vancomycin loaded spacers. The results do not favor Copal® spacem over Palacos® R+G for the use as a gentamicin and vancomycin biantibiotic-loaded spacer.

Antibiotic release and antibacterial efficacy in cement spacers and cement beads impregnated with different techniques: In vitro study.

OBJECTIVES: This study aims to compare the antibiotic release and biological effectiveness of bead type and articulating spacers of different cement types with antibiotics added at alternative phases of cement preparation. MATERIALS AND METHODS: Four gram vancomycin was added into two types of antibiotic-free cement (Simplex®, Biomet®) with similar viscosity and also gentamycin-containing cement (Refobacin®). Prepared specimens were used to create cement beads and articulating hip spacers, making a total of six different groups. Two alternative groups were formed by adding the Vancomycin while the cement was in dough phase. Antibiotic release and biological activity were evaluated with immunoassay techniques and agar-disk diffusion methods. RESULTS: All groups showed initial antibiotics surge in the first week, which was 2 to 4 times more evident in the beads group. Antibiotic release and change in release rate were significantly different between Simplex-alternative and Simplex, Biomet, Refobacin-beads, and between Biomet-spacer and Refobacin-beads groups (p<0.05). Elution of antibiotics was not different between mobile spacers prepared with conventional or alternative methods (p>0.05). Biomet cement showed larger diffusion inhibition zone in agar. There was no difference between biological activity of the bead and mobile designs of the Biomet brand (p>0.05). Inhibition zone analyses of agar and disk diffusion tests revealed significant differences between several groups (p<0.05). CONCLUSION: Cement beads provide superior antibiotic release regardless of cement type or preparation method. Simplex P® cement has lower anti-bacterial efficiency than Biomet®. Different methods for cement and antibiotics mixing and addition of extra vancomycin into the commercially drug loaded cement do not have any effect on the results.

Determination of four main components of gentamicin in animal tissues after solid-phase extraction by high-performance liquid chromatography/tandem mass spectrometry.

RATIONALE: An analytical method for gentamicin in animal tissues was developed and validated. An alkaline mobile phase with an HPH C8 column was selected so that all the four gentamicin components were retained and eluted without using fluorinated ion-pairing reagents. METHODS: The method is sufficiently sensitive and highly selective, using a strong cation-exchange solid-phase extraction cartridge (PCX) to clean up the samples. Different types of solid-phase extraction columns and membranes were considered to obtain a high recovery. The method was validated on spiking samples, recovery, inter- and intra-assay variation, to ensure its accuracy and precision. RESULTS: The LOQ (S/N ≥ 10) for gentamicin in goat meat, liver, kidney and adipose tissue was 25, 50, 30 and 30 ng/g, respectively; the LOD (S/N ≥ 3) was 5, 10, 10 and 10 ng/g, respectively. The recoveries were between 88% and 106%. The method in all animal tissues was calibrated from 10 to 1000 µg/L in the matrix-assisted standard solution. CONCLUSIONS: The novelty of this method is that the commonly used fluorinated ion-pairing reagent was not used in the mobile phase in our analysis, greatly reducing the contamination of the ESI source in negative mode. Moreover, the four gentamicin components were clearly separated via chromatographic separation.

Determination of gentamicin C components in fish tissues through SPE-Hypercarb-HPLC-MS/MS.

In this work, an HPLC/MS/MS method for determination of gentamicin C components in fish tissues was developed based on strong cation exchange solid-phase extraction (SPE) purification coupled with Hypercarb chromatographic column in separation mode. Sample was extracted using trichloroacetic acid aqueous solution containing EDTA. Ion-pairing reagents were not needed because of the "graphite polarity retention effect" of the Hypercarb chromatographic column. HPLC-MS/MS was performed in multiple reaction monitoring (MRM) mode for simultaneous qualitative and quantitative analyses (using matrix external standard) of gentamicin C components in fish tissues. Good linearity was obtained for the target analytes within the concentration range from 0.0100 to 0.500 mg/L. The limits of quantification (LOQ) of this method were 10.0, 20.0, and 20.0 µg/kg for C1, C1a, and sum of C2 + C2a, respectively. The average recoveries of gentamicin C components were 80.0%-110% when spiked at three levels with the blank carp (Cyprinus carpio) matrix, and the relative standard deviations (RSD) were all less than 15% (n = 6). In addition, for the features of simple operation, high sensitivity and good reproducibility, the proposed method has been successfully applied for detection of gentamicin residues in fish tissues during actual breeding.