Jette Tjørnelund

Determination of the distribution coefficient (logKd) of oxytetracycline, tylosin A, olaquindox and metronidazole in manure

Olaquindox (log Kow = -2.3) and metronidazole (log Kow = -0.1) both have low tendencies to sorp to particles in manure. This corresponds with the negative log Kow values of these antibiotics. Tylosin (log Kow = 1.63) and oxytetracycline (log Kow = -1.12) sorp relatively strongly to the manure particles and have log Kd values between 1.5 and 2.0. The tendency to bind to manure was ranked after increasing binding as follows: metronidazole < olaquindox << tylosin A and oxytetracycline. This order of ranking is consistent with results of sorption in soil. Our experiments illustrate that for some antibacterial agents estimation of the partitioning coefficients, Kd, cannot be made from Kow and f(oc) alone. Sorption of oxytetracycline to manure is much higher than expected from the negative log Kow value of the compound. It is believed that sorption of oxytetracycline to manure is influenced by ionic binding to divalent metal ions as such Mg2+ and Ca2+ as well as other charged compounds in the matrix. Binding of oxytetracycline to soil is stronger than the binding to manure. This is most likely due to the strong mineral related metal complexes formed between soil, metal ion and oxytetracycline. These complexes are not known to exist in manure. The relatively strong sorption of tylosin A to manure corresponds with data found for soil sorption of tylosin. Tylosin has a log Kow value of 2.5, thus it is not surprising that this drug binds strongly to manure.

Characterisation of the abiotic degradation pathways of oxytetracyclines in soil interstitial water using LC-MS-MS.

The fate of oxytetracyclines (OTCs) in soil interstitial water was investigated and the structure of a number of degradation products elucidated in a time-related experiment. A previously developed separation method for LC-MS-MS able to base separate and quantify OTC and three of its epimers and degradation products was applied. Compounds detected were 4-epi-oxytetracycline (EOTC) (t(R)=3.0 min), OTC (t(R)=4.4 min), alpha-apo-oxytetracycline (alpha-apo-OTC) (t(R)=11.4 min) and beta-apo-oxytetracycline (beta-apo-OTC) (t(R)=18.4 min). Furthermore, we tentatively identified 4-epi-N-desmethyl-oxytetracycline (E-N-DM-OTC) (t(R)=3.0 min), N-desmethyl-oxytetracycline (N-DM-OTC) (t(R)=3.5), N-didesmethyl-oxytetracycline (N-DDM-OTC), 4-epi-N-didesmethyl-oxytetracycline (E-N-DDM-OTC) (t(R)=3.7 and 4.7 min) and 2-acetyl-2-decarboxamido-oxytetracycline (t(R)=8.7) in all samples. Most compounds were only present in trace concentrations (less than 2%) relative to the parent OTC. EOTC was on the other hand formed up to a ratio of 0.6 relative to parent OTC concentration. Only EOTC, E-N-DM-OTC, N-DM-OTC, N-DDM-OTC and E-N-DDM-OTC were formed during the time-related experiment. All other compounds were probably only present as impurities in the spiked OTC formulation as they declined in concentration from the start of the experiment. Half-lives (T(1/2), days) of the OTCs in soil interstitial water were in the order of 2 days (EOTC) to 270 days (beta-apo-OTC).

Selectivity enhancement in capillary electrophoresis using non-aqueous media

Abstract Efficiency and selectivity are two important parameters in separation science in order to facilitate resolution of solutes. High efficiencies are easily obtained using capillary electrophoresis. Selectivity can be improved using different additives like surfactants or cyclodextrins. Recently, it has been shown that also non-aqueous capillary electrophoresis provides large improvements in selectivity and without the use of the above-mentioned additives.

Use of metal complexation in non-aqueous capillary electrophoresis systems for the separation and improved detection of tetracyclines.

Metal complexation in non-aqueous capillary electrophoresis systems was evaluated for the separation and improved detection of tetracycline antibiotics using laser-induced fluorescence detection. It was found that three factors were important for the choice of complexing agent: (i) it should be soluble in the organic solvent used for the separation, (ii) it should have a sufficient fast complexing rate so as not to invalidate the electrophoretic separation and, (iii) it should give a large increase in the fluorescence intensity. Mg2+ ions were found to be the most suitable ions for the separation of the tetracyclines as the acetate salt of magnesium is very soluble in organic solvents and only a relatively low current was generated during electrophoresis making it possible to use high concentrations of the complexing metal ion. Metal complexation strongly intensified the fluorescence of tetracyclines and all organic solvents investigated further intensified the fluorescence, e.g. dimethylformamide improved the fluorescence of the oxytetracycline metal complex by a factor of 34 compared to water. However, magnesium acetate was not sufficiently soluble in dimethylformamide and therefore N-methylformamide, improving the fluorescence intensity by only a factor of 9, was used. It was demonstrated that the method can be used for the detection of tetracyclines at the ppb level in milk and plasma.

Determination of oxytetracycline and its degradation products by high-performance liquid chromatography-tandem mass spectrometry in manure-containing anaerobic test systems

This paper describes the development of a HPLC-MS-MS (ESI) method with baseline separation of oxytetracycline, 4-epi-oxytetracycline, alpha-apo-oxytetracycline and beta-apo-oxytetracycline using an XTerra column and an MeOH-MilliQ-water (containing 8 mM formic acid) mobile phase. Limits of quantification for aqueous standards were in the range of 0.004 to 0.008 microM. The linear range tested was 0.003 to 0.5 microM and in one case up to 17 microM. An experiment simulating the degradation of oxytetracycline in manure was set up and free concentrations of the four antibiotics were determined during 6 months. Oxytetracycline (>0.02 microM) was observed up till 6 months after spiking. No important increase in free concentrations of the degradation products was observed.

DETERMINATION OF IMPURITIES IN TETRACYCLINE HYDROCHLORIDE BY NON AQUEOUS CAPILLARY ELECTROPHORESIS

Abstract A highly selective method for quantitative determination of impurities in tetracycline antibiotics based on non-aqueous capillary electrophoresis has been developed. In tetracycline hydrochloride the degradation products 4-epitetracycline, anhydrotetracycline and 4-epianhydrotetracycline can be determined with limits of detection corresponding to 0.06%, 0.04% and 0.02% of the drug substance, respectively. The relative standard deviations were about 4% at the 0.1% level of impurity in tetracycline hydrochloride. Furthermore, it is possible to detect desmethyltetracycline as well as a number of unknown impurities within 10 min. The separation of tetracycline, oxytetracycline, doxycycline and chlortetracycline by non-aqueous capillary electrophoresis is also demonstrated. The nature of the solvents and electrolytes in the electrophoresis medium exhibit a major influence on the separation selectivity. Solvents such as methanol and acetonitrile are more volatile than water and its is demonstrated that evaporation of the solvent from sample as well as from the electrophoresis medium may cause severe problems in some capillary electrophoresis instruments.

Chemical reactivity of the naproxen acyl glucuronide and the naproxen coenzyme A thioester towards bionucleophiles

Drugs may be metabolised to reactive electrophilic species that spontaneously react with proteins. The presence of such drug-protein adducts has been associated with drug toxicity. In this study, the reactivity of the major metabolite of naproxen--the 1-beta-O-glucuronide (Nap-GlcU)--was compared to the corresponding naproxen coenzyme A (Nap-CoA) thioester. The reactivity of the two metabolites was assessed in vitro in a phosphate buffer (pH 7.4; 0.1 M) at 37 degrees C towards the model bionucleophiles glutathione and human serum albumin (HSA). The reaction between the electrophilic species (Nap-GlcU and Nap-CoA) and glutathione forming the Nap-glutathione conjugate was monitored using LC-MS-MS and LC-UV, respectively. It was shown that Nap-CoA resulted in an approximate 100-fold higher formation of Nap-glutathione conjugate than Nap-GlcU. The presence of Nap-CoA also resulted in acylated HSA with a rate and a yield that was significantly higher than reported for Nap-GlcU. In summary, the data suggest that CoA metabolites may be more reactive species than acyl glucuronides that previously have been associated with severe drug related side effects in vivo.

Quantitative analysis of oxytetracycline and its impurities by LC-MS-MS

A liquid chromatographic-tandem mass spectrometric method using an Xterra MS C(18) chromatographic column ( 100 mm x 2.1 mm i.d., 3.5microm) that allows complete separation of oxytetracycline (OTC) and the impurities: 4-epi-oxytetracycline (EOTC), tetracycline (TC), 4-epi-tetracycline (ETC), 2-acetyl-2-decarboxamido-oxytetracycline (ADOTC), alpha-apo-oxytetracycline (alpha-AOTC) and beta-apo-oxytetracycline (beta-AOTC) was developed. Gradient elution was used and calibration curves were obtained using the scan mode selected reaction monitoring (SRM). Acceptable correlations were obtained for OTC, TC, EOTC and ADOTC whereas the correlations of alpha-AOTC and beta-AOTC were less accurate resulting in higher limits of quantification (LOQ) and limits of detection (LOD) relative to the other compounds. The intraday and interday accuracy varied for all the compounds from 90 to 112% and the intraday and interday precision were lower than 7.1%. The method was applied for analysis of commercial available ointments containing OTC resulting in an acceptable quantification of OTC and the impurities in the drug preparations. The advantage of this method compared to the other separation methods is an empty separation window right after the large peak corresponding to OTC in the chromatogram, which facilitates an accurate determination of ADOTC and the other impurities.

Non-aqueous capillary electrophoresis of drugs: properties and application of selected solvents.

The electrophoretic mobility of selected acidic and basic test solutes have been determined in non-aqueous media prepared by adding various combinations of ammonium acetate, sodium acetate, methane sulphonic acid and acetic acid to acetonitrile, propylene carbonate, methanol, formamide, N-methylformamide, N,N-dimethylformamide and dimethylsulphoxide, respectively. The apparent pH (pH*) of these non-aqueous media have been measured and it was found that pH* is an important factor for the separations in non-aqueous capillary electrophoresis. However, in some solvents the concentration of sodium acetate has a strong influence on the mobility despite very small changes in pH*. Due to the fact that a change in one parameter influences a number of other parameters it is very difficult to conduct systematic studies in non-aqueous media and to compare the migration of the species at fixed pH* values from one solvent to another. Thus pH* is only of value for comparison when used with a specific solvent or solvent mixture. The viscosity of the above-mentioned solvents were measured at various temperatures and means to adjust the viscosity of the non-aqueous media used for capillary electrophoresis are discussed and the separation of ibuprofen and its major metabolites in urine is used as an example.

Separation of neutral substances by non-aqueous capillary electrophoresis through interactions with cationic additives

Abstract The electrophoretic behaviour of neutral substances in non-aqueous capillary electrophoresis systems with tetraalkylammonium ions or long-chain trimethylammonium ions accompanied by different counter ions added have been investigated. Separation of neutral substances was only achieved in the highly dipolar aprotic solvents acetonitrile or propylene carbonate. The long-chain trimethylammonium ions were, however, not sufficiently soluble in acetonitrile. Thus, the studies comparing various types of ammonium ions were performed using propylene carbonate. The separation of the test solutes was improved with increasing concentrations of additives in the solvent. The type of ammonium ion as well as the counter ion were found to be important for the size of the separation window obtained. The separation principle was used for the determination of the water insoluble vitamin K 1 as well as the preservatives methylparaben and propylparaben in a pharmaceutical product.