Simultaneous separation and determination of active components in Cordyceps sinensis and Cordyceps militarris by LC/ESI-MS

A simple and rapid isocratic LC/MS coupled with electrospray ionization (ESI) method for simultaneous separation and determination of adenine, hypoxanthine, adenosine and cordycepin in Cordyceps sinensis (Cs) and its substitutes was developed. 2-Chloroadenosine was used as internal standard for this assay. The optimum separation for these analytes was achieved using the mixture of water, methanol and formic acid (85:14:1, v/v/v) as a mobile phase and a 2.0×150 mm Shimadzu VP-ODS column. Selective ion monitoring (SIM) mode ([M+H]⁺ at m/z 136, 137, 268, 252 and 302) was used for quantitative analysis of above four active components. The regression equations were liner in the range of 1.4–140.0 μg ml⁻¹ for adenine, 0.6–117.5 μg ml⁻¹ for hypoxanthine, 0.5–128.5 μg ml⁻¹ for adenosine and 0.5–131.5 μg ml⁻¹ for cordycepin. The limits of quantitation (LOQ) and detection (LOD) were, respectively 1.4 and 0.5 μg ml⁻¹ for adenine, 0.6 and 0.2 μg ml⁻¹ for hypoxanthine, 0.5 and 0.1 μg ml⁻¹ for adenosine and cordycepin. The recoveries of four constituents were from 93.5 to 107.0%. The nucleoside contents of various types of natural Cs and its substitutes were determined and compared with this developed method.     

In vitro distribution of ketoprofen enantiomers in articular tissues of osteoarthritic patients

The distribution of ketoprofen enantiomers in joint tissues was studied as a function of their relative tissular affinities using the multi-chamber distribution dialysis system described by Bickel et al. Selected off-cuts of synovial membrane, joint capsule, cartilage and ligament were obtained from ten patients suffering from osteoarthritis of the knee (n=3) or hip (n=7). Sörensen solution (4 ml) spiked with racemic ketoprofen (2 μg ml⁻¹) was dialysed against 1 ml of the four solutions of tissue homogenates (0.4 g ml⁻¹). Ketoprofen enantiomers were quantified in buffer and tissue solutions by high-performance liquid chromatography. The distribution of ketoprofen enantiomers in the Bickel's multi-compartment model indicated that there was a non-stereoselective affinity of ketoprofen enantiomers for their potential target tissues. Despite the interindividual variability in articular tissues, the concentrations (±S.D.) of R- and S-ketoprofen were significantly higher in synovial membrane (8.69 (4.76) μg g⁻¹ for S, 9.14 (5.57) μg g⁻¹ for R), joint capsule (5.71 (2.49) μg g⁻¹ for S, 5.49 (2.62) μg g⁻¹ for R) and ligament (6.28 (3.61) μg g⁻¹ for S, 6.40 (3.64) μg g⁻¹ for R) than in articular cartilage (3.67 (1.75) μg g⁻¹ for S, 3.70 (1.67) μg g⁻¹ for R). There were no significant differences in the distribution of R- and S-ketoprofen between the solutions of joint capsule, synovium and ligament tissues. These data may be related to differences in ketoprofen affinity for the different constituents of joints. This in vitro distribution profile is similar to that reported in vivo for other non-steroidal anti-inflammatory drugs.     

Simultaneous analysis of methylprednisolone, methylprednisolone succinate, and endogenous corticosterone in rat plasma

A reversed-phase HPLC method is reported for simultaneous quantitation of methylprednisolone (MP), MP succinate (MPS), and endogenous corticosterone (CST) in plasma of rats. Additionally, the 11-keto metabolite of MP (methylprednisone, MPN) is resolved from the other analytes. After addition of internal standard (triamcinolone acetonide; IS) and an initial clean up step, the analytes of interest are extracted into methylene chloride. The steroids are then resolved on a reversed-phase polymer column using a mobile phase of 0.1 M acetate buffer (pH 5.7): acetonitrile (77:23) which is pumped at a flow rate of 1.5 ml min⁻¹. Sample detection was accomplished using an UV detector at a wavelength of 250 nm. All the five components (MPS, MP, MPN, CST and IS) were baseline resolved from each other and other components of plasma. Linear relationships were found between the steroids: IS peak area ratios and plasma concentrations in the range of 0.1–4 μg ml⁻¹ for MP and MPS and 0.1–1.0 μg ml⁻¹ for MPN and CST. The assay is accurate as intra- and inter-run error values were <±8% for all the components. Further, the intra- and inter-run CVs of the assay were <16% at all the concentrations and for all the components. The application of the assay was demonstrated after the injection of a single 5 mg kg⁻¹ (MP equivalent) dose of MPS or a macromolecular prodrug of MP to rats.     

Spectrophotometric determination of pefloxacin in pharmaceutical preparations

It has been established that the antibiotic pefloxacin (Abaktal) methane-sulphonate reacts with Fe(III) at pH 1.00–8.00 to form a water-soluble complex with maximum absorbance at 360 nm. The composition of the complex, determined spectrophotometrically by the application of Job's, molar-ratio and Bent—French's methods, was pefloxacin: Fe(III) = 1:1 (pH = 2.50; λ = 360 nm; μ = 0.1 M). The relative stability constant, obtained by the methods of Sommer and Asmus was 10^5.02 (pH = 2.50; λ = 360 nm; μ = 0.1 M). The molar absorptivity of the complex at 360 nm was found to be 4.8 × 10³ l mol⁻¹ cm⁻¹, Beer's law was followed for pefloxacin concentrations of 2.15–85.88 μg ml⁻¹. The lower sensitivity limit of the method was 2.15 μg ml⁻¹. The relative standard deviation (n = 10) was 0.57–1.07%. The method can be applied to the rapid and simple determination of pefloxacin in aqueous solutions and tablets.     

Analysis of various nucleosides in plasma using solid phase extraction and high-performance liquid chromatography with UV detection

The National Cancer Institute (NCI) has screened many nucleosides for antiviral activity to the HIV-1 virus. Drugs demonstrating antiviral activity are tested in animal models to evaluate their toxicity and pharmacokinetic characteristics. These drugs are subsequently evaluated for efficacy in human clinical trials. Sensitive analytical methodology is needed to quantify nucleosides in plasma and other biological matrices in support of these studies. Battelle has modified and validated a reversed phase high-performance liquid chromatography (HPLC) method for several of these nucleosides that could be easily adapted for similar compounds. Methods have been validated for 6-chloro-2′,3′-dideoxyguanosine (6ClddG), 6-chloro-2′,3′-dideoxyinosine (6ClddI) and their primary metabolites 2′,3′-dideoxyguanosine (ddG) and 2′,3′-dideoxyinosine (ddI) in both rat and dog plasma containing EDTA. The method has also been validated for 2′-fluoro-2′,3′-dideoxyara-adenosine (βFlddA) and its primary metabolite 2′-β-fluorodideoxyinosine (βFddI) in rat plasma containing heparin. Calibration plasma standards were prepared over ranges of 0.1–10 μg ml⁻¹ for βFlddA and βFddI, 0.1–50 μg ml⁻¹ for 6ClddG and ddG, and 0.25–50 μg ml⁻¹ for 6ClddI and ddI in plasma containing 4 μg ml⁻¹ pentostatin. The addition of pentostatin to the plasma samples inhibits in-vitro deamination of the drug after collection. Quality control (QC) standards were prepared containing the appropriate anticoagulant and 4 μg ml⁻¹ pentostatin at concentrations within each of the bracketed calibration ranges in plasma. These methods have been successfully applied to plasma samples generated during various animal studies.     

A capillary GC-MS method for analysis of phenytoin and [C3]-phenytoin from plasma obtained from pulse dose pharmacokinetic studies

Stable isotope analogues of phenytoin are useful for pulse dose pharmacokinetic studies in epilepsy patients. A simultaneous assay was developed to quantitate phenytoin (5,5-diphenylhydantoin) and its stable isotope analogue [¹³C₃]-phenytoin (5,5-diphenyl-2,4,5-¹³C₃-hydantoin) from plasma. Quantitation was achieved by GC-MS analysis of liquid/liquid extracted plasma samples, with [²H₁₀]-phenytoin (5,5-di(pentadeuterophenyl)-hydantoin) as an internal standard. The total coefficients of variance (C.V._t) were <7% for phenytoin (2.5–40 μg ml⁻¹) and <10.3% for [¹³C₃]-phenytoin (0.1–6.0 μg ml⁻¹). The accuracy of the assay varied from 87.8–100.1% (phenytoin, 2.5–40 μg ml⁻¹) and 89.6–116.3% ([¹³C₃]-phenytoin, 0.02–6.0 μg ml⁻¹). The assay was tested under in vivo conditions by administration of a pulse dose of the stable isotope analogue to a single rat dosed to steady-state with fosphenytoin, a phenytoin prodrug. The results of the in vivo experiment demonstrate the usefulness of this assay for future pharmacokinetic studies in special population epilepsy patients.     

Simultaneous determination of valsartan and hydrochlorothiazide in tablets by first-derivative ultraviolet spectrophotometry and LC

First-derivative ultraviolet spectrophotometry and high-performance liquid chromatography (HPLC) were used to determine valsartan and hydrochlorothiazide simultaneously in combined pharmaceutical dosage forms. The derivative procedure was based on the linear relationship between the drug concentration and the first derivative amplitudes at 270.6 and 335 nm for valsartan and hydrochlorothiazide, respectively. The calibration graphs were linear in the range of 12.0–36.1 μg ml⁻¹ for valsartan and 4.0–12.1 μg ml⁻¹ for hydrochlorothiazide. Furthermore, a high- performance liquid chromatographic procedure with ultraviolet detection at 225 nm was developed for a comparison method. For the HPLC procedure, a reversed phase column with a mobile phase of 0.02 M phosphate buffer (pH 3.2)-acetonitrile (55: 45; v/v), was used to separate for valsartan and hydrochlorothiazide. The plot of peak area ratio of each drug to the internal standard versus the respective concentrations of valsartan and hydrochlorothiazide were found to be linear in the range of 0.06–1.8 and 0.07–0.5 μg ml⁻¹, respectively. The proposed methods were successfully applied to the determination of these drugs in laboratory-prepared mixtures and commercial tablets.     

Pharmacokinetics of thrombin-like enzyme from venom of Agkistrodon halys ussuriensis Emelianov determined by ELISA in the rat.

A thrombin-like enzyme (TLE) was separated and purified from the venom of a northeast Chinese snake Agkistrodon halys ussuriensis Emelianov. Experiments were performed in rats to determine the pharmacokinetic parameters following an intravenous (i.v.) or a subcutaneous (s.c.) injection of the thrombin-like enzyme. The plasma levels of TLE were estimated by enzyme-linked immunosorbent assay. The method exhibited high reproducibility and accuracy in correlating optical densities with TLE concentrations (0.2–30 ng ml⁻¹, r=0.99). The plasma concentration-time course after i.v. administration of 50 μg kg⁻¹ TLE was well fitted by a two-compartment open model. The half-life of the -phase was 18.0±3.2 min, and that of the β-phase 3.9±0.7 h. The apparent volume of distribution was 1.8±0.5 l kg⁻¹, and clearance was 5.4±0.5 ml min⁻¹ kg⁻¹. When the TLE was injected s.c. at a dose of 0.75 mg kg⁻¹, the changes in plasma concentration were best described by a two-compartment model with a first-order absorption. The maximal plasma level of 51±2.7 ng ml⁻¹ was reached at 5.2±0.5 h. The absorption rate constant was 0.3±0.03 h⁻¹. The area under the plasma concentration-time curve (AUC) was 2.8±0.8 μg h⁻¹ ml⁻¹.     

Flow-injection biamperometric determination of epinephrine

A flow-injection manifold is proposed for the determination of epinephrine. The experimental procedure is based on the indirect biamperometric detection of the drug by using Fe(III)-Fe(II) as an indicating redox system and a flow-through detector with two polarized Pt wire electrodes. The calibration graph is linear over the range 0.3–20 μg ml⁻¹ of epinephrine. The relative standard deviation for the determination of 10 μg ml⁻¹ of epinephrine is 1.5% (n = 25) and the sample throughput is 153 h⁻¹. The method was applied to the determination of epinephrine in two commercially available pharmaceutical preparations.     

Simultaneous LC determination of paracetamol and related compounds in pharmaceutical formulations using a carbon-based column

A simple, rapid and convenient high performance liquid chromatographic method, which permits the simultaneous determination of paracetamol, 4-aminophenol and 4-chloracetanilide in pharmaceutical preparation has been developed. The chromatographic separation was achieved on porous graphitized carbon (PGC) column using an isocratic mixture of 80/20 (v/v) acetonitrile/0.05 M potassium phosphate buffer (pH 5.5) and ultraviolet detection at 244 nm. Correlation coefficient for calibration curves in the ranges 1–50 μg ml⁻¹ for paracetamol and 5–40 μg ml⁻¹ for 4-aminophenol and 4-chloroacetanilide were >0.99. The sensitivity of detection is 0.1 μg ml⁻¹ for paracetamol and 0.5 μg ml⁻¹ for 4-aminophenol and 4-chloroacetanilide. The proposed liquid chromatographic method was successfully applied to the analysis of commercially available paracetamol dosage forms with recoveries of 98–103%. It is suggested that the proposed method should be used for routine quality control and dosage form assay of paracetamol in pharmaceutical preparations. The chromatographic behaviour of the three compounds was examined under variable mobile phase compositions and pH, the results revealed that selectivity was dependent on the organic solvent and pH used. The retention selectivity of these compounds on PGC was compared with those of octadecylsilica (ODS) packing materials in reversed phase liquid chromatography. The ODS column gave little separation for the degradation product (4-aminophenol) from paracetamol, whereas PGC column provides better separation in much shorter time.     

Use of palladium(II) chloride as colour-forming reagent in determination of pralidoxime chloride in water and tablets

Pralidoxime chloride (PAM-2Cl) has been determined spectrophotometrically in Britton—Robinson buffer solution at pH = 6.45; the method is based on measurement of the absorbance of the Pd(II)-pralidoxime complex at 327 nm. Studies of the composition of the complex by Job's continuous variation method, the molar ratio method and Bent—French's method yielded a Pd(II):pralidoxime ratio of 1:1. The conditional stability constant (K′) of the complex at the optimum pH of 6.45 and an ionic strength (μ) of 0.3 M was found to be 10^5.2. The molar absorptivity was 1.05 × 10⁴ 1 mol⁻¹ cm⁻¹. Beer's law was obeyed at concentrations up to 60 μM. The detection limit was 0.55 μg ml⁻¹. The relative standard deviation (N = 10) was 0.28–1.03%. The method was accurate and sensitive for the analysis of PAM-2Cl in water and tablets.     

Spectrophotometric determination of oxytetracycline in pharmaceutical preparations using sodium molybdate as analytical reagent

A spectrophotometric method is proposed for the determination of oxytetracycline in pharmaceutical preparations. The method is based on the measurement of the absorbance of the molybdate—oxytetracycline complex at 404 nm (pH 5.50; μ = 0.1 M; 20°C). The composition of the complex (1:1) was determined by the application of the spectrophotometric methods of Job and Bent—French (pH 5.50; λ = 390 nm; μ = 0.1 M). The relative stability constant (K′ = 10^4.6) of the complex was obtained by the methods of Sommer and Nash (pH 5.50; λ = 390 nm; μ = 0.1 M; 20°C). The molar absorptivity of the complex was 9.5 × 10³ l mol⁻¹ cm⁻¹. Beer's law was obeyed over the concentration range 2.48–34.78 μg ml⁻¹. The relative standard deviation RSD (n = 10) was 0.27–0.39%. The method proposed can be applied to the assay of oxytetracycline in capsules. The detection limit of oxytetracycline is 2.5 μg ml⁻¹.     

Spectrophotometric determination of alendronate in pharmaceutical formulations via complex formation with Fe(III) ions

The formation of the complex between alendronate, non-chromophoric bisphosphonate drug important for the treatment of a variety of bone diseases, and iron(III) chloride in perchloric acid solution was studied. The stoichiometric ratio of alendronate to Fe(III) ions in the chromophoric complex was determined to be 1:1. The conditional stability constant was log K′_ave=4.50 (SD=0.15), indicating that the Fe(III)–alendronate complex is a complex of medium stability. The optimum conditions for this reaction were ascertained and a spectrophotometric method was developed for the determination of alendronate in the concentration range 8.1–162.5 μg ml⁻¹, the detection limit being 2 μg ml⁻¹. The method was validated for the direct determination of alendronate in tablet dosage formulations.     

Stimulation of gastric alkaline secretion by stable prostacyclin analogues in rat and dog

The stimulation of gastric alkaline secretion in vivo following topical mucosal application of four stable anti-ulcer analogues of prostacyclin has been investigated in the rat and in the dog using intragstric pH-stat techniques. In the rat, basal alkaline secretion was significantly stimulated by the prostacyclin analogues 16-phenoxy-(5)-5,9-epoxy-PGF₁ (16-phenoxy) and its methyl ester, when administered in the luminal perfusion fluid. The 16-phenoxy analogue (25 and 50 μg ml⁻¹) increased basal alkaline secretion with its methyl ester being more potent. The methyl ester of 16,16-dimethyl PGI₁ (25 and 50 μg ml⁻¹) likewise stimulated alkaline secretion whereas its corresponding free acid was inactive at these concentrations. In further studies in the conscious dog with a Heidenhain gastric pouch, these four prostacyclin analogues administered intraluminally (1.25 μg ml⁻¹) significantly increased gastric alkaline secretion. The present findings indicate that the stable 16-phenoxy and 16,16-dimethyl analogues of prostacyclin, can stimulate gastric alkaline secretion in the dog and rat in vivo. As with the previously reported 16,16-dimethyl PGE₂ analogue, this property of stimulating alkaline secretion may therefore contribute to the antiulcer activity of these prostacyclin analogues.     

Spectrophotometric and atomic absorption spectrometric determination of certain cephalosporins

Two sensitive spectrophotometric and atomic absorption spectrometric procedures are developed for the determination of certain cephalosporins (cefotaxime sodium and cefuroxime sodium). The spectrophotometric methods are based on the charge-transfer complex formation between these drugs as n-donors and 7,7,8,8-tetracyano-quinodimethane (TCNQ) or p-chloranilic acid (p-CA) as π-acceptors to give highly coloured complex species. The coloured products are measured spectrophotometrically at 838 and 529 nm for TCNQ and p-CA, respectively. Beer’s law is obeyed in a concentration range of 7.6–15.2 and 7.1–20.0 μg ml⁻¹ with TCNQ, 95.0-427.5 and 89.0-400.5 μg ml⁻¹ with p-CA for cefotaxime sodium and cefuroxime sodium, respectively. The atomic absorption spectrometric methods are based on the reaction of the above cited drugs after their alkali-hydrolysis with silver nitrate or lead acetate in neutral aqueous medium. The formed precipitates are quantitatively determined directly or indirectly through the silver or lead content of the precipitate formed or the residual unreacted metal in the filtrate by atomic absorption spectroscopy. The optimum conditions for hydrolysis and precipitation have been carefully studied. Beer’s law is obeyed in a concentration range of 1.9–11.4 and 1.78–8.90 μg ml⁻¹ with Ag(I), 14.2–57.0 and 13.3–53.4 μg ml⁻¹ with Pb(II) for cefotaxime sodium and cefuroxime sodium, respectively (for both direct and indirect procedures). The spectrophotometric and the atomic absorption spectrometric procedures hold well their accuracy and precision when applied to the analysis of cefotaxime sodium and cefuroxime sodium dosage forms.     

Development and validation of a reversed-phase liquid chromatographic method for the assay of lidocaine in aqueous humour samples

A simple, fast and reliable reversed-phase liquid chromatographic method was developed for the assay of lidocaine in human aqueous humour samples. The samples were analysed without any preliminary treatment on a C8 column with UV detection at 225 nm. The mobile phase consisted of methanol/sodium dihydrogen phosphate (30 mM) containing sodium pentansulphonate (10 mM) adjusted to pH 2.5 with phosphoric acid (50:50 v/v). Validation of the method showed it to be precise, accurate and linear over the concentration range of analysis with a limit of detection of 0.2 μg ml⁻¹. The limit of quantitation was 2.5 μg ml⁻¹ with a relative standard deviation of 2.5%. Linear regression analysis in the range 2.5–60 μg ml⁻¹ gave correlation coefficients higher than 0.999. No interference from three commonly co-administered drugs was observed. The method developed was applied to the analysis of lidocaine in aqueous humour samples in order to evaluate and compare the efficacy of two different forms of administration of lidocaine for topical anaesthesia in cataract surgery.     

Simultaneous determination of fosinopril and hydrochlorothiazide in pharmaceutical formulations by spectrophotometric methods

Three new spectrophotometric procedures for the simultaneous determination of fosinopril and hydrochlorothiazide are described. The first method, derivative-differential spectrophotometry, comprised of measurement of the difference absorptivities derivatized in the first-order (ΔD₁) of a tablet extract in 0.1 N NaOH relative to that of an equimolar solution in methanol at wavelengths of 227.6 and 276.4 nm, respectively. The second method, depends on the application ratio spectra derivative spectrophotometric method to resolve the interferance due to spectral overlapping. The analytical signals were measured at 237.9, 243.8 nm for fosinopril and 262.4, 269.3 and 278.6 nm for hydrochlorothiazide in the binary mixture, in the first derivative of the ratio spectra of the mixture solutions in methanol. Calibration graphs were established for 4.0–50.0 μg ml⁻¹ fosinopril and 2.0–14.0 μg ml⁻¹ hydrochlorothiazide in binary mixture. The third method, absorbance ratio method, the determination of fosinopril and hydrochlorothiazide was performed by using the absorbances read at 210.0, 219.5 and 271.7 nm in the zero-order spectra of their mixture. The developed methods were compared with absorbance ratio method. Application of the suggested procedures were successfully applied to the determination of this compound in synthetic mixtures and in pharmaceutical preparations, with high percentage of recovery, good accuracy and precision.     

Determination of meloxicam in bulk and pharmaceutical formulations

Three sensitive and reproducible methods for quantitative determination of meloxicam (mel) in pure form and in pharmaceutical formulations are presented. The first method is high performance liquid chromatography by which the drug is determined in the presence of its degradation products over concentration range 100–500 μg ml⁻¹ with mean percentage accuracy 100.13±0.53. The second method is based on measuring the absorbance of the formed neutral complex between basic methylene blue and mel in phosphate buffer (pH 8) at λ=653.5 nm over concentration range 1–5 μg ml⁻¹ with mean percentage accuracy 99.12±1.18. The third method is based on reaction between 2,3-dichloro-5,6-dicyano-p-benzoquinone resulting in the formation of an intense orange red coloured product after heating in a boiling water bath for 5 min. The coloured product exhibits an absorption maximum at 455 nm, over concentration range 40–160 μg ml⁻¹ with mean percentage accuracy 100.53±1.04.     

Development and validation of a novel LC non-derivatization method for the determination of amikacin in pharmaceuticals based on evaporative light scattering detection

A novel method for the direct determination of the aminoglycoside antibiotic amikacin and its precursor component kanamycin was developed and validated, based on reversed phase LC with evaporative light scattering detector (ELSD). ELSD response to amikacin was found to be enhanced by: (a) use of ion-pairing acidic reagents of increased molecular mass, (b) increase of mobile phase volatility and (c) decrease of peak width and asymmetry (obtained by controlling the mobile phase acidity and/or ratio of organic solvent to water). Utilizing a Thermo Hypersil BetaBasic C₁₈ column, the selected optimized mobile phase was water–methanol (60:40, v/v), containing 3.0 ml l⁻¹ nonafluoropentanoic acid (18.2 mM) (isocratic elution with flow rate of 1.0 ml min⁻¹). ELSD experimental parameters were: nitrogen pressure 3.5 bar, evaporation temperature 50 °C, and gain 11. Amikacin was eluted at 8.6 min and kanamycin at 10.4 min with a resolution of 1.5. Logarithmic calibration curves were obtained from 7 to 77 μg ml⁻¹ (r > 0.9995) for amikacin and 8 to 105 μg ml⁻¹ (r > 0.998) for kanamycin, with a LOD equal to 2.2 and 2.5 μg ml⁻¹, respectively.

In amikacin sulfate pharmaceutical raw materials, the simultaneous determination of sulfate (t_R = 2.3 min, LOD = 1.8 μg ml⁻¹, range 5–40 μg ml⁻¹, %R.S.D. = 1.1, r > 0.9997), kanamycin and amikacin was feasible. No significant difference was found between the results of the developed LC–ELSD method and those of reference methods, while the mean recovery of kanamycin from spiked samples (0.5%, w/w) was 97.3% (%R.S.D. ≤ 2.0, n = 6). Further, the developed method was applied for the determination of amikacin in pharmaceutical formulations (injection solutions) without any interference from the matrix (recovery from spiked samples ranged from 95.6 to 103.8%).     

Determination of cisapride, its oxidation product, propyl and butyl parabens in pharmaceutical dosage form by reversed-phase liquid chromatography

A simple, rapid and reproducible high-performance liquid chromatography (HPLC) assay for cisapride, its oxidation product (OP), propyl and butyl parabens in a pharmaceutical formulation is described. Chromatography was performed at room temperature by pumping acetonitrile–20 mM phosphate buffer pH 7 (50:50, v/v) at 1.5 ml min⁻¹ through C₈ reversed-phase column. Cisapride, OP, propyl and butyl parabens were detected at 276 nm and were eluted at 9.7, 3.1, 5.1 and 7.1 min, respectively. Calibration plots were linear (r>0.999) for all compounds from 0.5 to 200 μg ml⁻¹ for cisapride and OP and 0.1–200 μg ml⁻¹ for propyl and butyl parabens. Detection limits for cisapride, OP, propyl and butyl parabens were 40, 46, 48 and 54 ng ml⁻¹, respectively. Forced degradation investigations showed that cisapride does not undergo degradation under heat, acidic and basic conditions but it was susceptible to oxidation. The proposed method was successfully applied to the assay of cisapride in the presence of preservatives and OP in a commercial suspension.