Determination of cycloserine in microdialysis samples using liquid chromatography-tandem mass spectrometry with benzoyl chloride derivatization.

A new method for the analysis of cycloserine (4-amino-3-isoxazolidinone, CYC) in rat microdialysis samples has been developed. This method consists of derivatizing the CYC with benzoyl chloride, which transforms primary amines into highly stable derivatives. An attractive feature of this method was that the derivatization reaction is straightforward and can be completed within 10 min. The formed derivative, in contrast to the non-derivatized analyte, exhibited increased chromatographic retention and decreased matrix effects resulting from the co-elution of other components using reversed-phase liquid chromatography and on-line switching. Detection on a quadrupole-linear ion trap mass spectrometer (AB3200 Q-Trap) was performed using electrospray tandem mass spectrometry in multiple reaction monitoring mode. Various derivatization parameters were optimized in order to improve chromatographic separation and minimize ion suppression. In particular, the benzoylation reaction was improved to enhance the reproducibility and sensitivity of the chromatographic method. The transition m/z 207.1 → 105.1 was acquired to monitor the CYC derivatization products. The method was fully validated for its sensitivity, selectivity, matrix effect and stability. A good linearity over the selected range (r > 0.99, range = 22-2200 mg/L), as well as accuracy and precision within ±7% of the target values, was obtained. The assay described herein was successfully applied to quantitatively measure CYC in the lung and blood of anesthetized rats.

Estimation of content of anti-TB drugs supplied at centres of the Revised National TB Control Programme in Tamil Nadu, India.

OBJECTIVE: To determine the content of certain antituberculosis (TB) drugs supplied at TB treatment centres of the Revised National TB Control Programme (RNTCP) in the state of Tamil Nadu, India. METHODS: Eight districts across the state were selected, and the following drugs were collected from five settings (District TB centre, TB unit, designated microscopy centres, DOT providers) in each district: rifampicin (150 and 450 mg), isoniazid (300 mg), pyrazinamide (500 and 750 mg), ethambutol (400 and 600 mg), ethionamide (250 mg), levofloxacin (500 mg) and cycloserine (250 mg). A maximum of 10 tablets/capsules were collected from each setting. The drugs were coded prior to analysis. All drugs were assayed by validated spectrophotometric methods. The acceptable limits for drug content were taken as 90-110% of the stated content. RESULTS: More than 90% of tablets of rifampicin 450 mg, isoniazid 300 mg, pyrazinamide 500 and 750 mg, ethambutol 400 and 600 mg and ethionamide 250 mg were within acceptable limits. Eighty per cent of rifampicin 150 mg, 21% of cycloserine 250 mg and 87% of levofloxacin 500 mg were within acceptable limits. The mean cycloserine content was below the acceptable limit in all districts, the mean drug content being 200 mg (range: 108-245 mg). CONCLUSION: This systematic study showed that the stated drug content of cycloserine was not reached in all districts. Deterioration of cycloserine could be minimised by storing the drug in refrigerators. The geographical location of the districts had no influence on the drug content.

Development of a liquid chromatographic method for the determination of related substances and assay of d-cycloserine.

d-cycloserine or d-4-amino-3-isoxazolidinone is an antibiotic produced by Streptomyces garyphalus and Streptomyces orchidaceus. d-Cycloserine is used in the second line treatment of tuberculosis and is often used in developing countries. Therefore, expensive high-tech techniques are not recommended for analysis. Here, a liquid chromatography method with ultraviolet detection (LC-UV) is described using a base deactivated column (Hypersil BDS column; 25 cm x 4.6 mm I.D.) kept at 45 degrees C. The gradient method uses mobile phases containing acetonitrile (ACN), 20mM sodium octane sulphonate (SOS), 0.2M potassium dihydrogen phosphate buffer pH 2.8, water: A: (4:70:10:16v/v/v/v) and B: (17:70:10:3v/v/v/v). The method proved to be robust, linear, repeatable, sensitive, selective and easy to perform. For the related substances test 50 microl of a 0.5 mg/ml d-cycloserine solution is injected. For assay, a concentration of 0.1 mg/ml is proposed to avoid overloading of the detector.

The interaction of cycloserine with pyruvate and other biologically relevant alpha-ketoacids.

The ability of cycloserine solutions to deplete alpha-oxoacids has been found to be correlated with the spontaneous transformation of cycloserine into a derivative dimer (2,5-bis-(aminoxymethyl)-3,6-diketopiperazine). Synthetic dimer was found to react rapidly with pyruvate to form the expected oxime. Two lines of evidence indicate that it is the cycloserine dimer and not cycloserine itself that reacts with alpha-ketoacid. First, the 1H NMR spectrum of the purified oxime is superimposable with that arising when the dimer and pyruvate are mixed and the spectrum taken immediately thereafter. Second, the mass spectrum of the reaction product of cycloserine dimer and methylpyruvate is totally consistent with the formation of a stable oxime derivative. Furthermore, when cycloserine is incubated with pyruvate the oxime derived from the dimer is found. These observations clearly indicate that cycloserine in solution can have chemical activities in addition to its ability to interfere with pyridoxal dependent reactions. On these grounds it is concluded that any biological action of cycloserine should be interpreted cautiously.

Spectrophotometric determination of cycloserine with 9-methoxyacridine.

Spectrophotometric assay for cycloserine based on the interaction of the drug with 9-methoxyacridine as a chromogenic agent is described. The highly colored substituted acridine product was identified as 9-(d-4-imino-3-isoxazolidinone)acridine. Color development was affected by time and temperature of heating and by the quantity of 9-methoxyacridine reagent utilized. The absorbance at 438 nm is linearly proportional to concentrations of cycloserine with a detection limit of 0.3 microgram/mL. The optimum range for the assay of cycloserine was from 5.0 X 10(-6) to 3.0 X 10(-4) M (correlation coefficient = 0.9999, n = 6). When applied to cycloserine capsules labeled to contain 250 mg, the proposed method gave mean recoveries of 101.84 +/- 0.48%. The procedure is sufficiently sensitive, precise, and accurate for the determination of cycloserine in its dosage form.

Role of the solvent on the stability of cycloserine under ESI-MS conditions.

The effects of methanol (M) and acetonitrile (A) on the stability of cycloserine (1) have been studied. InfraRed Multiphoton PhotoDissociation (IRMPD) spectroscopy of the ionic species from electrospray ionization tandem mass spectrometry (ESI-MS) of 1/M and 1/A solutions points to extensive dimerization of 1 to cis-3,6-bis(aminooxymethyl)-2,5-piperidinedione (2), while the same process is not observed in the ESI-MS of 1/M solutions. 1D and 2D nuclear magnetic resonance experiments confirmed these findings by showing that partial dimerization of 1 actually takes place at room temperature in acetonitrile even before ESI-MS analysis. Comparison of nuclear magnetic resonance and IRMPD spectroscopic data from the same 1/A solution suggests that dimerization of cycloserine is enhanced in the ESI source.

Comparison of ChromID C. difficile agar and cycloserine-cefoxitin-fructose agar for the recovery of Clostridium difficile.

AIM: The rapidly changing epidemiology of Clostridium difficile infection highlights the need for improved and continuing surveillance involving stool culturing to enable molecular tracking. Culture of C. difficile can be difficult and time consuming. In this report ChromID C. difficile agar (CDIF) was compared to cycloserine-cefoxitin-fructose-egg-yolk agar which contained 0.1% sodium taurocholate (TCCFA) as a germinant. RESULTS: All ribotypes of C. difficile tested (n=90) grew well on CDIF within 24 h and most gave characteristic small irregular black colonies with a raised umbonate profile. Counts from standard suspensions of C. difficile at 24 h (p<0.005) and 48 h (p=0.01) were significantly higher on CDIF than on TCCFA. Similar results were achieved after alcohol shock. When temperature shock was used to differentiate vegetative cells and spores, the total number of culturable and vegetative cells on CDIF was significantly higher than on TCCFA (culturable cells, p=0.003 at 24 h and p=0.002 at 48 h; vegetative cells, p=0.0003 at 24 h and p=0.0002 at 48 h). CONCLUSIONS: These data suggest that CDIF is a better medium for the recovery of vegetative C. difficile than TCCFA and equal to TCCFA for spore recovery.

Analysis of cycloserine and related compounds using aqueous normal phase chromatography/mass spectrometry.

A new approach is evaluated for the analysis of cycloserine, a strongly hydrophilic drug. The method utilized is aqueous normal phase chromatography with a silica hydride-based stationary phase and mass spectrometry for detection. The samples are analyzed to determine the number of components and they are identified when possible. In addition, the composition change is monitored with respect to time and sample solvent. Analyses using both gradient and isocratic conditions are presented. The repeatability of inter- and intraday analyses is also determined.

Development and validation of indirect RP-HPLC method for enantiomeric purity determination of D-cycloserine drug substance.

A new chiral purity method was developed for D-cycloserine (D-cys) by reverse phase HPLC and validated. Chiral derivatizing reagents, viz., o-phthalaldehyde and N-acetyl-L-cysteine were utilized in this method. The resultant diastereomers were resolved using Zorbax SB Phenyl HPLC column under isocratic elution. A mobile phase of 95:05 (v/v), 20mM Na(2)HPO(4) (pH 7), and acetonitrile, respectively, was used with the flow rate of 1.0 mL/min and UV detection at 335 nm. The method development with different chiral stationary phases and chiral derivatization reagents were also investigated. The stability of diastereomer derivative and influence of organic modifier and pH of the mobile phase were studied and optimized. The stability-indicating capability of the method was established by performing stress studies under acidic, basic, oxidation, light, humidity and thermal conditions. The detection and quantitation limit of L-cycloserine (L-cys) were 0.015 and 0.05% (w/w), respectively. A linear range from 0.05 to 0.30% (w/w) was obtained with the coefficient of determination (r(2)) 0.998. The recovery obtained for L-cys was between 92.9 and 100.2%. This method was applied successfully in pharmaceutical analysis to determine the content of L-cys in D-cys bulk drug.

Simultaneous quantification of cycloserine and its prodrug acetylacetonylcycloserine in plasma and urine by high-performance liquid chromatography using ultraviolet absorbance and fluorescence after post-column derivatization.

D-Cycloserine is a broad-spectrum antibiotic used with other antibiotics to treat various forms of tuberculosis. Its prodrug sodium (R)-4-[(1-methyl-3-oxo-1-butenyl)amino]-3-isoxazolidinone hemihydrate, developed for better aqueous stability and solubility, is combined with another broad-spectrum antibiotic, fludalanine. An ion-pair, reversed-phase high-performance liquid chromatographic assay has been developed to simultaneously detect cycloserine and its prodrug in plasma and urine. The prodrug is detected directly by ultraviolet absorbance and cycloserine by fluorescence following post-column derivatization.

Tripeptide hydroxamate from Corynebacterium kutscheri.

A tripeptide hydroxamate was isolated from cultures of Corynebacterium kutscheri grown on an iron-limiting medium. The metabolite was characterized by spectral measurements and by chemical degradation as L-alpha-aspartyl-L-alpha-N-hydroxy-aspartyl-D-cycloserine.

Chromatographic analysis of antituberculosis drugs in biological samples.

Numerous chromatographic and non-chromatographic methods of analysis for anti-tuberculosis drugs and metabolites in biological tissues have been discussed in this review. Depending upon the analytical methodology selected, limits of detection range from microgram to picogram levels. A number of examples have been given of the correlation between different types of assay procedures. The metabolism and pharmacokinetics have been described along with some of the commonly associated problems of sample collection and storage.