Development and Validation of an HPLC Method for Determination of Ceftiofur Sodium

An isocratic high-performance liquid chromatographic method has been developed for assay of ceftiofur sodium in drug substance and in sterile powder for injection. Chromatography was performed on a 250 mm × 4.6 mm, 5 μm particle, C₁₈ column with a 78:22 (v/v) mixture of 0.02 m disodium hydrogen phosphate buffer (pH adjusted to 6.0 with 85% orthophosphoric acid) and acetonitrile as mobile phase, at a flow rate of 1.0 mL min⁻¹. The separation was monitored by UV detection at 292 nm. Validation of the method for linearity and range, intra- and inter-day precision, accuracy, specificity, recovery, robustness, and limits of quantification and detection yielded good results. The calibration plot was linear from 20.0–120.0 μg mL⁻¹ and the correlation coefficient was 0.9999. It was shown that ceftiofur was degraded under acidic, alkaline, oxidative, and photolytic conditions. The method was found to be stability-indicating and could be used for routine analysis of ceftiofur sodium for injection.     

Application of a Validated, Stability-Indicating LC Method to Stress Degradation Studies of Ramipril and Moexipril.HCl

A stability-indicating reversed-phase liquid chromatographic (RPLC) method has been established for analysis of ramipril (RAM) and moexipril hydrochloride (MOEX.HCl) in the presence of the degradation products generated in studies of forced decomposition. The drug substances were subjected to stress by hydrolysis (0.1 m NaOH and 0.1 m HCl), oxidation (30% H₂O₂), photolysis (254 nm), and thermal treatment (80 °C). The drugs were degraded under basic and acidic conditions and by thermal treatment but were stable under other stress conditions investigated. Successful separation of the drugs from the degradation products was achieved on a cyanopropyl column with 40:60 (v/v) aqueous 0.01 m ammonium acetate buffer (pH 6)–methanol as mobile phase at a flow rate of 1 mL min⁻¹. Detection was by UV absorption at 210 nm. Response was a linear function of concentration over the range 5–50 μg mL⁻¹ (r > 0.9995), with limits of detection and quantitation (LOD and LOQ) of 0.04 and 0.09 μg mL⁻¹, respectively, for RAM and 0.014 and 0.32 μg mL⁻¹, respectively, for moexipril. The method was validated for specificity, selectivity, solution stability, accuracy, and precision. Statistical analysis proved the method enabled reproducible and selective quantification of RAM and MOEX as the bulk drug and in pharmaceutical preparations. Because the method effectively separates the drugs from their degradation products, it can be used as stability-indicating.     

Determination of (E)-3,5,4′-Trimethoxystilbene in Rat Plasma by LC with ESI-MS

(E)-3,5,4′-trimethoxystilbene (BTM-0512) is a resveratrol analog with a variety of pharmacological action, including anti-cancer properties, anti-allergic activity, estrogenic activity, antiangiogenic activity, and vascular-targeting activity against microtubule-destabilization. There is, however, no validated analytical method for quantification of (E)-3,5,4′-trimethoxystilbene in biological matrices, so pharmacokinetic data and suitable methods for determination of the compound in plasma are currently lacking. A rapid and sensitive liquid chromatographic–mass spectrometric method for determination of (E)-3,5,4′-trimethoxystilbene in rat plasma, using carbamazepine as internal standard, has been developed and validated. Plasma samples were treated with acetonitrile to precipitate proteins. Samples were then analyzed by HPLC on a 250mm × 4.6 mm i.d., 5-μm particle, C₁₈ column with methanol–water, 80:20 (v/v), containing 10 mm ammonium acetate and 0.2% formic acid (pH 3.0), as mobile phase, delivered at 0.85 mL min⁻¹. A single-quadrupole mass spectrometer with an electrospray interface operated in selected-ion monitoring mode was used to detect [M + H]⁺ ions at m/z 271.3 for (E)-3,5,4′-trimethoxystilbene and m/z 237.5 for the internal standard. (E)-3,5,4′-trimethoxystilbene and the internal standard eluted as sharp, symmetrical peaks with retention times of 8.9 and 4 min, respectively. Calibration plots for (E)-3,5,4′-trimethoxystilbene in rat plasma at concentrations ranging from 0.01 to 5.0 μg mL⁻¹ were highly linear. Intra-day and inter-day precision, as RSD, was <12.9%, and accuracy was in the range 94.8–104.7%. The limit of detection in plasma was 0.005 μg mL⁻¹. The method was successfully used to determine the concentration of (E)-3,5,4′-trimethoxystilbene after oral administration of 86 mg kg⁻¹ of the drug to Sprague–Dawley rats and can be used to investigate the pharmacokinetics of the compound.     

Liquid chromatographic determination of erythromycins in fermentation broth

Summary A simple and sensitive isocratic LC method is described for the determination of erythromycins in fermentation broths. A simple technique utilizing acetone-methyl ethyl ketone, 1∶1, as extraction solvent was coupled with suitable chromatographic conditions—compounds were separated on a 250 mm×4.6 mm i.d., 5 μm, reversed-phase column at 65°C with acetonitrile-0.2m K₂HPO₄ pH7.0-water, 35:5:60 (v/v), as mobile phase at a flow rate of 1.0 mL min⁻¹. UV detection was performed at 215 nm. Separation of erythromycin F from polar components of the fermentation liquid was sufficient. Erythromycins A, B, C, D, and E, andN-desmethylerythromycin A were also separated, as were known decomposition products of erythromycin A and several unknown components. The method is suitable for monitoring the progress of erythromycin fermentation.     

Determination and Pharmacokinetic Study of Calycosin-7-O-β-d-glucoside in Rat Plasma After Intravenous Administration of Aidi Lyophilizer

Aidi injection is a clinical medicine used in China for the treatment of cancer. Calycosin-7-O-β-d-glucoside is the main effective components of the formulas. In this study, a high performance liquid chromatographic (LC) method was developed to quantify calycosin-7-O-β-d-glucoside in rat plasma using a liquid–liquid extraction and ultraviolet (UV) absorbance detection. LC analysis was performed on a Diamonsil C₁₈ column (200 × 4.6 mm i.d., 5 μm particle size) with isocratic mobile phase consisting of acetonitrile–0.05% phosphoric acid (19.5:80.5, v/v) of a flow rate of 1.0 mL min⁻¹. The linear range was 0.11–17.6 μg mL⁻¹ and the low quantification limit was 0.11 μg mL⁻¹ (S/N = 10). The intra- and inter-day relative standard deviations (RSD) in the measurement of quality control (QC) samples 0.11, 0.22, 1.32 and 8.80 μg mL⁻¹ ranged from 4.1 to 6.3 and 4.3 to 6.2%, respectively. The accuracy was from −6.7 to 4.3% in terms of relative error (RE). Calycosin-7-O-β-d-glucoside was stable in storage at −20 °C for 2 weeks and stable after three freeze–thaw cycles in rat plasma. This method was validated for specificity, accuracy, precision and was successfully applied to pharmacokinetic study of calycosin-7-O-β-d-glucoside in rat plasma after intravenous administration of Aidi lyophilizer.     

Determination of the antibiotic zinc bacitracin in animal food by high-performance liquid chromatography with ultraviolet detection

Summary A high-performance liquid chromatographic method with ultraviolet detection has been developed for the analysis of the polypeptide antibiotic zinc bacitracin in adulterated animal feed. Firstly, the process for extraction of the antibiotic from the feed was optimized. This process involved extraction from the feed at pH 2, centrifugation, liquid-liquid extraction, then solidphase extraction. The extract obtained was then dissolved in the mobile phase and injected into the chromatograph. The best analytical results were obtained by use of a C₁₈ column with a mobile phase comprising a 50:50 (%,v/v) mixture of 0.3m phosphate buffer, pH 3, containing 20mm sodium dodecyl sulfate (SDS) and 19:1 (v/v) acetonitrile-methanol. The analytical signal (peak area) was monitored at 254 nm. The calibration function was estimated between 200 and 1000 mg L⁻¹. The proposed method was applied to the analysis of zinc bacitracin present in different fortified animal feed products at levels between 5 and 200 mg kg⁻¹. Recovery rates were between 66 and 85% and the relative standard deviation was below 7%.     

New cobalt and iron pivalate complexes with 2,2′:6′,2″-terpyridine: synthesis, structure, and magnetic properties

The mononuclear complexes (η³-terpy)M(Piv)²·MeCN (M = Fe ⁱⁱ (3) and Co ⁱⁱ (4), and Piv is the pivalate anion) were synthesized by the reactions of polymeric iron(ii) and cobalt(ii) pivalates with 2,2′:6′,2″-terpyridine (terpy). The oxidation of compound 3 affords the pentanuclear heterospin iron(ii,iii) complex (η³-terpy)Fe₅(μ₄-O)(μ₃-OH)(μ-OH)₂(μ-Piv)₇(η¹-Piv)₂ (5). All compounds were characterized by X-ray diffraction. Dedicated to the 90th anniversary of the L. Ya. Karpov Institute of Physical Chemistry. Published in Russian in Izvestiya Akademii Nauk. Seriya Khimicheskaya, No. 6, pp. 1186–1190, June, 2008.     

LC-MS-MS Determination of Cyclo{(2S)-2-amino-8-[(aminocarbonyl)hydrazono] decanoyl-1-l-tryptophyl-l-isoleucyl-(2R)-2-piperidinecarbonyl} a Novel Histone Deacetylase Inhibitor in Rat Serum

A rapid and sensitive liquid chromatography-tandem mass spectrometry assay was developed for the determination of a novel histone deacetylase inhibitor, cyclo{(2S)-2-amino-8-[(aminocarbonyl)hydrazono]decanoyl-1-l-tryptophyl-l-isoleucyl-(2R)-2-piperidinecarbonyl} (SD-2007), in rat serum. The mobile phase consisted of acetonitrile and ammonium formate (10 mM) (85:15 v/v), and the flow rate was 0.25 mL min⁻¹. Chromatographic separations were achieved by isocratic elution on a C₁₈ column. Multiple reaction monitoring was based on the transition of m/z = 681.8 → 83.6 for SD-2007 and 372.1 → 176.1 for trazodone (internal standard). A linearity was observed over a concentration range from 2 to 1,000 ng mL⁻¹ (r ² > 0.999), with the lower limit of quantification at 2 ng mL⁻¹ with 100 μL of rat serum. The mean intra- and inter-day assay accuracy ranged from 98.5–109.7% to 95.2–102.7%, respectively, and the mean intra- and inter-day precision was between 4.3–11.3% and 2.9–13.3%. The developed assay was applied to a pharmacokinetic study of SD-2007 in rats after intravenous injection (dose 4 mg kg⁻¹).     

Triphenyltetrazolium chloride as a new analytical reagent for molybdenum(VI): Application to plant analysis

Solvent extraction of molybdenum(VI) ion associate with triphenyltetrazolium chloride (TTC) has been studied. TTC was proposed as reagent for the spectrophotometric determination of micro amounts of molybdenum(VI) at λ_max 250 nm. The optimum conditions for extraction of molybdenum(VI) as an ionassociation complex with TTC has been determined. Beer’s law is obeyed in the range of 0.5–10 μg/mL molybdenum(VI). The molar absorptivity of the ion-pair is 1 × 10⁶ L/mol cm. The sensitivity of the method is 9.6 × 10⁻⁵ μg/cm². The characteristic values for the extraction equilibrium and the equilibrium in the aqueous phase are: distribution constant K _D = 32.64, extraction constant K _ex = 2.19 × 10¹⁰ association constant β = 6.71 × 10⁸. The interferences of different cations, anions on molybdenum(VI) determination were also investigated. A sensitive and selective method for the determination of microquantities of molybdenum(VI) has been developed. The determination was carried out without preliminary separation of molybdenum. A novel procedure of molybdenum(VI) extraction and spectrophotometric determination in different plant samples was examined.     

Development of a Validated Stability-Indicating LC Method for Nitazoxanide in Pharmaceutical Formulations

A reversed-phase liquid chromatographic (LC) method was developed for the assay of nitazoxanide (NTZ) in solid dosage formulations. An isocratic LC separation was performed on a Phenomenex Synergi Fusion C₁₈ column (250 mm × 4.6 mm, i.d., 4 μm particle size) using a mobile phase of 0.1% o-phosphoric acid solution, pH 6.0: acetonitrile (45:55, v/v) at a flow rate of 1.0 mL min⁻¹. Detection was achieved with a photodiode array detector at 240 nm. The detector response for NTZ was linear over the concentration range from 2 to 100 μg mL⁻¹ (r = 0.9999). The specificity and stability-indicating capability of the method were proved using stress conditions. The RSD values for intra-day precision were less than 1.0% for tablets and powder for oral suspension. The RSD values for inter-day precision were 0.6 and 0.7% for tablets and powder for oral suspension. The accuracy was 100.4% (RSD = 1.8%) for tablets and 100.9% (RSD = 0.3%) for powder for oral suspension. The limits of quantitation and detection were 0.4 and 0.1 μg mL⁻¹. There was no interference of the excipients on the determination of the active pharmaceutical ingredient. The proposed method was precise, accurate, specific, and sensitive. It can be applied to the quantitative determination of drug in tablets and powder for oral suspension.     

Molecularly imprinted polymer based on chemiluminescence imaging for the chiral recognition of dansyl-phenylalanine

A new molecularly imprinted polymer (MIP)–chemiluminescence (CL) imaging detection approach towards chiral recognition of dansyl-phenylalanine (Phe) is presented. The polymer microspheres were synthesized using precipitation polymerization with dansyl-l-Phe as template. Polymer microspheres were immobilized in microtiter plates (96 wells) using poly(vinyl alcohol) (PVA) as glue. The analyte was selectively adsorbed on the MIP microspheres. After washing, the bound fraction was quantified based on peroxyoxalate chemiluminescence (PO-CL) analysis. In the presence of dansyl-Phe, bis(2,4,6-trichlorophenyl)oxalate (TCPO) reacted with hydrogen peroxide (H₂O₂) to emit chemiluminescence. The signal was detected and quantified with a highly sensitive cooled charge-coupled device (CCD). Influencing factors were investigated and optimized in detail. Control experiments using capillary electrophoresis showed that there was no significant difference between the proposed method and the control method at a confidence level of 95%. The method can perform 96 independent measurements simultaneously in 30 min and the limits of detection (LODs) for dansyl-l-Phe and dansyl-d-Phe were 0.025 μmol L⁻¹ and 0.075 μmol L⁻¹ (3σ), respectively. The relative standard deviation (RSD) for 11 parallel measurements of dansyl-l-Phe (0.78 μmol L⁻¹) was 8%. The results show that MIP-based CL imaging can become a useful analytical technology for quick chiral recognition.     

Simultaneous RP-LC Determination of Losartan Potassium, Ramipril, and Hydrochlorothiazide in Pharmaceutical Preparations

A simple, rapid, and precise reversed-phase high-performance liquid chromatographic method has been developed for simultaneous determination of losartan potassium, ramipril, and hydrochlorothiazide. The three drugs were separated on a 150 mm × 4.6 mm i.d., 5 μm particle, Cosmosil C₁₈ column. The mobile phase was 0.025 m sodium perchlorate–acetonitrile, 62:38 (v/v), containing 0.1% heptanesulphonic acid, pH adjusted to 2.85 with orthophosphoric acid, at a flow rate of 1.0 mL min⁻¹. UV detection was performed at 215 nm. The method was validated for linearity, accuracy, precision, and limit of quantitation. Linearity, accuracy, and precision were acceptable in the ranges 35–65 μg mL⁻¹ for losartan, 1.75–3.25 μg mL⁻¹ for ramipril, and 8.75–16.25 μg mL⁻¹ for hydrochlorothiazide.     

Simultaneous Determination of Seven Active Compounds in Radix Salviae Miltiorrhizae by Temperature-Controlled Ultrasound-Assisted Extraction and HPLC

A simple and effective high-performance liquid chromatographic (HPLC) method has been developed for simultaneous quantification of three phenolic acids (3,4-dihydroxyphenyllactic acid (Chinese name danshensu), protocatechuic aldehyde, and salvianolic acid B) and four diterpenes (dihydrotanshinone I, cryptotanshinone, tanshinone I, and tanshinone II_A) in radix salviae miltiorrhizae. Chromatography was performed on a 250 mm × 4.6 mm i.d., 5-μm particle size, C₁₈ column. The mobile phase was a linear gradient prepared from 0.1% (v/v) aqueous formic acid and acetonitrile at a flow-rate of 1.0 mL min⁻¹. All the target components were well separated with high resolution and without interference. Good linearity (R ² > 0.999) was observed over the concentration ranges investigated, and intra-day and inter-day precision were high. Temperature-controlled ultrasound-assisted extraction was used to prevent hydrolysis of thermally unstable components during the sample-extraction procedure, and the extraction conditions were carefully optimized. Recovery of the seven components was from 98.45 to 100.63% and relative standard deviations were always <1.5%. The validated method was successfully used for simultaneous quantification of the three phenolic acids and the four diterpenes in radix salviae miltiorrhizae of different geographic origins.     

Bioconversion of <Emphasis Type="SmallCaps">d</Emphasis>-glucose into <Emphasis Type="SmallCaps">d</Emphasis>-glucosone by Glucose 2-oxidase from <Emphasis Type="Italic">Coriolus versicolor</Emphasis> at Moderate Pressures

Glucose 2-oxidase (pyranose oxidase, pyranose:oxygen-2-oxidoreductase, EC 1.1.3.10) from Coriolus versicolor catalyses the oxidation of d-glucose at carbon 2 in the presence of molecular O₂ producing d-glucosone (2-keto-glucose and d-arabino-2-hexosulose) and H₂O₂. It was used to convert d-glucose into d-glucosone at moderate pressures (i.e. up to 150 bar) with compressed air in a modified commercial batch reactor. Several parameters affecting biocatalysis at moderate pressures were investigated as follows: pressure, [enzyme], [glucose], pH, temperature, nature of fluid and the presence of catalase. Glucose 2-oxidase was purified by immobilized metal affinity chromatography on epoxy-activated Sepharose 6B-IDA-Cu(II) column at pH 6.0. The rate of bioconversion of d-glucose increased with the pressure since an increase in the pressure with compressed air resulted in higher rates of conversion. On the other hand, the presence of catalase increased the rate of reaction which strongly suggests that H₂O₂ acted as inhibitor for this reaction. The rate of bioconversion of d-glucose by glucose 2-oxidase in the presence of either nitrogen or supercritical CO₂ at 110 bar was very low compared with the use of compressed air at the same pressure. The optimum temperature (55°C) and pH (5.0) of d-glucose bioconversion as well as kinetic parameters for this enzyme were determined under moderate pressure. The activation energy (E _a) was 32.08 kJ mol⁻¹ and kinetic parameters (V _max, K _m, K _cat and K _cat/K _m) for this bioconversion were 8.8 U mg⁻¹ protein, 2.95 mM, 30.81 s⁻¹ and 10,444.06 s⁻¹ M⁻¹, respectively. The biomass of C. versicolor as well as the cell-free extract containing glucose 2-oxidase activity were also useful for bioconversion of d-glucose at moderate pressures. The enzyme was apparently stable at moderate pressures since such pressures did not affect significantly the enzyme activity.     

Production and Physico-chemical Characterization of a Biosurfactant Produced by <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> OBP1 Isolated from Petroleum Sludge

Pseudomonas aeruginosa strain OBP1, isolated from petroleum sludge, was used to produce biosurfactant from a modified mineral salt medium with 2% n-hexadecane as sole source of carbon. The crude biosurfactant was fractionated using TLC and HPLC. Using FTIR spectroscopy, ¹H NMR, and LC-MS analyses, the chemical structure of the purified fraction of crude biosurfactant was identified as rhamnolipid species. The LC-MS spectra show that monorhamnolipid (l-rhamnopyranosyl-β-hydroxydecanoyl-β- hydroxydecanoate, Rha-C₁₀-C₁₀) was produced in abundance with the predominant congener [M–H]⁻ ions for l-rhamnopyranosyl-l-rhamnopyranosyl-3-hydroxydecanoyl-3-hydroxydecanoate (Rha-Rha-C₁₀-C₁₀). Seven different carbon substrates and five nitrogen sources were examined for their effect on rhamnolipid production. Using n-hexadecane (20 g/l) as carbon substrate and urea along with (NH₄)₂SO₄ (2 g/l each) as nitrogen source was found to be the best, with a maximum yield of 4.8 g/l. The biosurfactant reduced the surface tension of water to 31.1 mNm⁻¹ with a critical micelle concentration of 45 mg/l. The biosurfactant showed a better emulsifying activity against a variety of hydrocarbon and achieved a maximum emulsion index of 82% for diesel. The purified biosurfactant showed a significant antibacterial activity against Staphylococcus aureus at a minimum inhibitory concentration of 8 μg/ml.     

A Temperature and Salt-Tolerant <Emphasis Type="SmallCaps">l</Emphasis>-Glutaminase from Gangotri Region of Uttarakhand Himalaya: Enzyme Purification and Characterization

Purification and characterization of halotolerant, thermostable alkaline l-glutaminase from a Bacillus sp. LKG-01 (MTCC 10401), isolated from Gangotri region of Uttarakhand Himalaya, is being reported in this paper. Enzyme has been purified 49-fold from cell-free extract with 25% recovery (specific activity 584.2 U/mg protein) by (NH₄)₂SO₄ precipitation followed by anion exchange chromatography and gel filtration. Enzyme has a molecular weight of 66 kDa. l-Glutaminase is most active at pH 11.0 and stable in the pH range 8.0–11.0. Temperature optimum is 70 °C and is completely stable after 3 h pre-incubation at 50 °C. Enzyme reflects more enhanced activity with 1–20% (w/v) NaCl, which is further reduced to 80% when NaCl concentration was increased up to 25%. l-Glutaminase is almost active with K⁺, Zn²⁺, and Ni²⁺ ions and K _m and V _max values of 240 μM and 277.77 ± 1.1 U/mg proteins, respectively. Higher specific activity, purification fold, better halo-tolerance, and thermostability would make this enzyme more attractive for food fermentation with respect to other soil microbe derived l-glutaminase reported so far.     

Sequential and simultaneous statistical optimization by dynamic design of experiment for peptide overexpression in recombinant Escherichia coli

The production of recombinant anti-HIV peptide, T-20, in Escherichia coli was optimized by statistical experimental designs (successive designs with multifators) such as 2^4–1 fractional factorial, 2³ full factorial, and 2² rotational central composite design in order. The effects of media compositions (glucose, NPK sources, MgSO₄, and trace elements), induction level, induction timing (optical density at induction process), and induction duration (culture time after induction) on T-20 production were studied by using a statistical response surface method. A series of iterative experimental designs was employed to determine optimal fermentation conditions (media and process factors). Optimal ranges characterized by %T-20 (proportion of pepttide to the total cell protein) were observed, narrowed down, and further investigated to determine the optimal combination of culture conditions, which was as follows: 9, 6, 10, and 1 mL of glucose, NPK sources, MgSO₄, and trace elements, respectively, in a total of 100 mL of medium inducted at an OD of 0.55–0.75 with 0.7 mM isopropyl-β-d-thiogalactopyranoside in an induction duration of 4 h. Under these conditions, up to 14% of T-20 was obtained. This statistical optimization allowed, the production of T-20 to be increased more than twofold (from 6 to 14%) within, a shorter induction duration (from 6 to 4 h) at the shake-flask scale. Coauthors.     

Polysaccharides of Fabaceae. VI. Galactomannans from seeds of <Emphasis Type="Italic">Astragalus alpinus</Emphasis> and <Emphasis Type="Italic">A. tibetanus</Emphasis>

Galactomannans with galactose:mannose ratios 1:1.48 and 1:1.33, [α]_D +67.9 and +76.4°, [η] 870.3 and 1337.1 mL/g, and molecular weights 999 and 1549 kDa, respectively, were isolated in 0.59 and 4.65% yields (of seed mass) from seeds of Astragalus alpinus and A. tibetanus (Fabaceae). Physicochemical methods (CrO₃ oxidation; methylation–GC/MS; IR, NMR, and ¹³C spectroscopy) found that the main polysaccharide chain consisted of 1,4-β-D-mannopyranose units substituted 67.5% (A. alpinus) and 75.2% (A. tibetanus) at the C-6 position by single α -D-galactopyranose units. The contents of mannobiose blocks Man–Man, (Gal)Man–Man/Man–Man(Gal), and (Gal)Man–Man(Gal) variously substituted with galactose were according to ¹³C NMR spectroscopy 15.9, 55.5, and 28.6% in A. alpinus galactomannan and 9.9, 42.3, and 47.8% in A. tibetanus galactomannan.     

Analysis of spectinomycin in fermentation broth by reversed-phase chromatography

A pre-column derivatized high-performance liquid chromatographic (HPLC) method with ultraviolet-visible detection was developed to measure the concentrations of spectinomycin in fermentation broth. Derivatization reagents, 2,4-dinitrophenylhydrazine in acetonitrile (5 mg mL⁻¹) and trifluoroacetic acid in acetonitrile (0.8 mol L⁻¹), were added to an aliquot of the fermentation broth, and the mixture was incubated for 60 min at 70°C. The resulting derivative was separated from other compounds by isocratic elution in a reversed-phase column Zorbax SB-C18 (250 mm × 4.6 mm, 5 μm). Mobile phase consisted of acetonitrile, tetrahydrofuran, and water (φ _r = 40: 35: 25) and the flow rate was 1.0 mL min⁻¹. The detection wavelength was 415 nm. The standard curve for spectinomycin sulfate was linear with correlation coefficients of 0.9997 in the range of 25 μg mL⁻¹ to 600 μg mL⁻¹. The relative standard deviation values ranged from 0.43 % to 2.18 % depending on the concentration of samples. The average recovery was 101.5 %. The limit of detection was 50 ng mL⁻¹.