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 high pH based reversed-phase high performance liquid chromatographic method for the analysis of aminoglycoside plazomicin and its impurities.

A reversed-phase high performance liquid chromatographic (RP-HPLC) method has been developed for the aminoglycoside (AG) plazomicin (ACHN-490). This method employed a high pH mobile phase (pH>11) with a gradient of 0.25 M ammonium hydroxide in water and acetonitrile, an XBridge C(18) column and UV detection at 210 nm. Although the molar UV absorption of plazomicin is weak, the high pH conditions of this method allow for higher loadings, which compensates for the inherent low UV sensitivity. Under these high pH conditions, impurities and degradants were base line separated from plazomicin. The mobile phases used for this method allowed for on-line mass detection for the impurities and degradants. The RP-HPLC method has been validated in terms of specificity, linearity and range, accuracy, and precision. The analytical method met specificity requirements of a homogenous peak with no interferences from the blank or from the known impurities in plazomicin. The linearity of the method for the plazomicin impurity determination was excellent, with a coefficient of determination (r(2)) of 0.9993, over the freebase (FB) concentration range of 0.0025-3.0 mg/mL. The method is capable of detecting impurities down to 0.1% of the peak area of plazomicin. A single point standard at a concentration of 1.0 mg/mL FB was validated over the range of 50-150% for quantitation of the freebase content (the assay) in bulk drug substance. The mean recoveries of FB are in the range 98.6-102.0% with a mean RSD (relative standard deviation) <1.0%. The study also examined the method precision for purity, impurities and the assay with two instruments on two different days. The method showed adequate accuracy and precision for the intended use. This high pH method was successfully used to determine the impurity and measure the drug content in the final plazomicin drug substance. In addition, the method with an on-line mass spectrometry detector has been used to characterize the structures of the impurities in plazomicin.

Immunoassays for the rapid detection of gentamicin and micronomicin in swine muscle.

A monoclonal antibody (mAb)-based ELISA and strip test for gentamicin (GEN) and its analogue micronomicin (MIN), are reported in this study. The conjugate gentamicin-glutaraldehyde-bovine serum albumin (GEN-GDA-BSA) was used as an immunogen. The produced anti-GEN mAB exhibited high cross-reactivity with micronomicin (MIN; 131.2%) and slight or negligible crossreactivity with other aminoglycosides. Based on this mAB, an ELISA and a strip test for GEN and MIN were developed and evaluated. The ELISA showed a 50% inhibition concentration (IC50) of 0.75 ng/mL for GEN and 0.58 ng/mL for MIN. For GEN, the average recoveries at 25-200 microg/kg ranged from 73 to 91%, with intraday CVs of 9-16% and interday CVs of 8-15%. For MIN, the average recoveries ranged from 108 to 131%, with intraday CVs of 10-16% and interday CVs of 8-15%. In contrast, the strip test for GEN or MIN had a detection limit of 5 ng/mL in phosphate-buffered saline and 50 microg/kg in muscle (n=24), and the results could be judged within 10 min. The detection results of incurred samples analyzed by the strip test, ELISA, and HPLC indicated that the two immunoassays correlated well with the HPLC method and could be used as convenient tools for the rapid screening of GEN and MIN residues in swine muscle.

Micellar electrokinetic chromatography of aminoglycosides.

The components of the aminoglycosides, e.g., gentamicin, sisomicin, netilmicin, kanamycin, amikacin, and tobramycin, and related impurities of these antibiotics can be separated by means of micellar electrokinetic chromatography (MEKC). Derivatization with o-phthaldialdehyde and thioglycolic acid is found to be appropriate for all antibiotics. The background electrolyte was composed of sodium tetraborate (100 mM), sodium deoxycholate (20 mM), and beta-cyclodextrin (15 mM) and has a pH value of 10.0. This method is valid for evaluation of gentamicin, kanamycin, and tobramycin. It has yet to be adopted for amikacin, paramomycin, neomycin, and netilmicin.

Improved liquid chromatographic method with pulsed electrochemical detection for the analysis of gentamicin.

The official method for the determination of the composition and related substances of gentamicin prescribed by the European Pharmacopoeia (Ph. Eur.) is liquid chromatography combined with pulsed electrochemical detection (LC-PED). However, this method utilizes a polymer stationary phase which shows rather low efficiency towards the separation of the main gentamicin components. Moreover, the mobile phase contains a lot of non volatile salts: sodium sulphate and sodium octanesulphonate. Following a comparative study, the most performant LC-PED method has been evaluated and validated using a reversed phase C18 column (C18, 250 x 4.6mm ID, 110 A, 5 microm) kept at 35 degrees C with a mobile phase containing volatile ion pairing agents: trifluoroacetic acetic acid (TFA) and pentafluoropropionic acid (PFPA). In addition to the selectivity of the main gentamicin components and its related substances, the method is repeatable, linear and proves to be robust. It is also applicable to a wider number of C18 columns.

A new micellar electrokinetic capillary chromatography method for separation of the components of the aminoglycoside antibiotics.

Aminoglycoside antibiotics are always a mixture of structurally related amino sugars, which do not have a chromophore or fluorophore. The aim of the study was to find one method for evaluation of the components and impurities of the antibiotics. Derivatization with o-phthaldialdehyde and thioglycolic acid is found to be appropriate for all antibiotics. The components of gentamicin (GM), sisomicin, netilmicin, kanamycin, amikacin, and tobramycin were tried to separate by means of micellar electrokinetic chromatography. The background electrolyte was composed of sodium tetraborate (100 mM, pH 10.0), sodium deoxycholate (20 mM), and beta-cyclodextrin (15 mM). This method is valid for evaluation of GM, kanamycin, and tobramycin. It has to be improved for amikacin and netilmicin. In addition, 46 bulk samples of GM of different manufacturer or pharmaceutical companies were investigated. Many samples were found to contain many minor products and different amounts. Beside different patterns of the main compounds GM C1, GM C1a, GM C2a, and GM C2, many lots were found consisting of a substantial number of minor products. The appearance of a high number of minor products is always associated with the existence of sisomicin, which is not found in "pure" samples. However, almost all samples met the requirements of the European Pharmacopoeia (EP) and United States Pharmacopoeia (USP).

Sensitive high-performance liquid chromatographic assay for aminoglycosides in biological matrices enables the direct estimation of bacterial drug uptake.

Following the development of a sensitive high-performance liquid chromatographic (HPLC) assay for gentamicin in biological matrices, the utility of this assay for the determination of other clinically important aminoglycosides (neomycin, netilmicin and sisomicin) in bacterial culture media or plasma is demonstrated. The high sensitivity of the assay enables direct measurement of the aminoglycoside content of bacterial cells cultured in the presence of unlabelled drug.

[Spectrophotometric determination of amikacin and sisomicin in the air using the Hantzsch reaction]. / Spektrofotometricheskii metod opredeleniia amikatsina i sizomitsina v vozdukhe s ipol'zovaniem reaktsii Gancha.

A procedure for determination of amikacin and sisomicin in ambient air of production areas was developed. It is based on the Hantzsch reaction involving formation of dihydrolutidine derivatives on the antibiotic interaction with acetyl acetone and formaldehyde in acid media. The yellow-colored reaction products are subjected to spectrophotometry at 356 nm. The detection limits for amikacin and sisomicin are 0.005 and 0.01 mg respectively. The procedure may be used for routine sanitary inspection of ambient air for every of the above antibiotics in the absence of the other in production of the substances and their dosage forms.

Intraocular penetration of sisomicin in rabbits.

The intraocular penetration of sisomicin, an aminoglycoside antibiotic, was studied in 47 normal rabbits following subconjunctival and/or intramuscular (IM) injections. Sisomicin levels were determined in the cornea and the aqueous humor of the injected eye as well as in the aqueous of the fellow eye by means of the cylinder-plate bioassay technique. When the IM route alone was used, the eye tissue concentrations remained negligible. One hour after subconjunctival injection, highly therapeutic sisomicin concentrations were achieved in the aqueous humor of both injected and fellow eyes, but the level dropped to very low concentrations 12 hours after the injection. The concomitant administration of sisomicin intramuscularly and subconjunctivally resulted in a significant delay of the clearance of sisomicin from the eye tissues. The use of the combined subconjunctival-IM regimen for administering aminoglycoside antibiotics needs further evaluation.

[Pharmacokinetic investigations on sisomicin in children (author's transl)]. / Pharmakokinetische Untersuchungen von Sisomicin unter besonderer Berücksichtigung des frühen Kindesalters.

Sisomicin is an aminoglycoside effective against gramnegative germs. The sensitiveness of coli, proteus, pseudomonas and klebsiella ranges from 0,1 to 0,4 mcg/ml. Germs with inhibition-values of up to 1 mcg/ml are certainly Sisomicin-sensitive. The side-effects of Sisomicin resemble those of other aminoglycosides, as for instance lesions of the VIIth cranial nerve and the kidney. Since aminoglycosides have a relatively small therapeutic range between toxicity and effective minimal-concentration, investigations are important especially in children. 66 children of different age groups received Sisomicin in doses of 3 mg/kg, 2 mg/kg and 1 mg/kg, and examined pharmacokineticly. Serum-levels were measured after 30 min 1, 2, and 4h, in some cases also after 6 and 8 h. In a separate group we determined the values after 5, 10, 15, 20, 30 and 40 min. The urinary output was controlled and the content of Sisomicin in meconium determined. Based on these results we recommend an individual, Sisomicin dosage for each age group. Clinically the Sisomicin proved to be well tolerated and effective antibioticum.

Radioimmunoassay and radioenzymatic assay of a new aminoglycoside antibiotic, netilmicin.

A radioimmunoassay and a radioenzymatic assay for netilmicin, a new aminoglycoside, were developed in our laboratories to assist in the study of the pharmacology of the drug and to establish values for use in its monitoring. The assays are sensitive, precise, and rapid, giving results that correlate (r = 0.90) with each other and with those of a microbiological assay in which Klebsiella pneumoniae is used as the test organism. Preliminary pharmacological studies show the drug to have a biological half-life of 135 min. which is comparable to that for other aminoglycosides.

Comparative distribution of gentamicin, tobramycin, sisomicin, netilmicin, and amikacin in interstitial fluid in rabbits.

We compared the penetration of five aminoglycosides into interstitial fluid (IF). IF was obtained in rabbits from Silastic tissue cages. Intramuscular injections were made: 1.5 mg/kg per dose for gentamicin (G), tobramycin (T), sisomicin (S), and netilmicin (N) and 7.5 mg/kg per dose for amikacin (A). Serum levels and IF concentrations were studied for 12 h after a single injection. IF levels were also compared in a six-injection study (one injection every 8 h). Peak serum levels were significantly higher with A than with G, T, S, and N, which gave similar concentrations. In IF, G gave the highest levels 1 h after the first injection. At 4 and 8 h, the concentrations achieved with G and A were similar but significantly greater than those achieved with T, S, and N. Twelve hours after a single injection, N gave higher IF levels than the other drugs except A. In the six-injection study, the IF levels of G and A reached 4.6 +/- 1.5 and 5.27 +/- 1.1 microgram/ml, respectively, at 48 h. S and N gave identical concentrations (2.07 +/- 0.25 and 2.42 +/- 0.42 microgram/ml, respectively). T induced the lowest levels (1.17 +/- 0.30 microgram/ml). Thus, in this rabbit model, the IF concentrations achieved with G and A were above the minimal inhibitory concentrations for most susceptible strains. Possible relations between IF aminoglycoside concentrations and therapeutic efficiency or toxicity are pointed out but deserve further studies.

The structure of antibiotic G-52, a new aminocyclitol-aminoglycoside antibiotic produced by Micromonospora zionensis.

Antibiotic G-52, a new aminocyclitol-aminoglycoside antibiotic produced in the fermentation of Micromonospora zionensis, has been shown to be 6'-N-methylsisomicin on the basis of its spectral characteristics. This assignment was confirmed by synthesis of the antibiotic from sisomicin.

Radioimmunoassay of sisomicin.

An antibody induced in rabbits against a gentamicin-bovine serum albumin conjugate cross-reacted with the chemically related aminoglycoside, sisomicin, facilitating the development of a sisomicin radioimmunoassay. Sisomicin was labeled with (125)I using an acylating agent [3-(4-hydroxyphenyl)propionic acid-N-hydroxy-succinimide ester]. The resulting assay produced a linear standard curve on a logit-log plot with a sensitivity of 140 pg. Comparison with a microbiological assay showed no significant difference (P < 0.001) in the measurement of sisomicin by these two methods.