Comparative Study Between Zero-Order Spectra-Processing and Ratio Spectra-Manipulating Methods Applied for the Determination of Isoxsuprine Hydrochloride in the Presence of Its Oxidative Degradation Product.

Four accurate, precise, and validated stability-indicating spectrophotometric methods handling either zero-order spectra or ratio spectra have been developed and compared for the analysis of isoxsuprine hydrochloride (ISX) in the presence of its oxidative degradation product. The first two methods processed zero-order spectra, namely graphical absorbance ratio or Q-Analysis and area under the curve, whereas the third and fourth methods manipulated ratio spectra, namely the ratio difference spectrophotometric method and derivative ratio. The proposed methods showed good linearity in the range of 2-23 µg/mL. The methods were tested for specificity using laboratory-prepared mixtures containing the drug and its degradation product. The proposed methods were applied for the determination of ISX in Vascular tablets and the obtained results were acceptable, with small percentage RSD values. The validity of the proposed procedures was further assessed by applying the standard addition technique, which showed no interference from excipients. The obtained results were statistically compared with those obtained by the reported method, showing no significant differences when t- and F-tests were applied.

Spectrofluorimetric determination of certain adrenergic agonist drugs in their pure forms and pharmaceutical formulations: Content uniformity test application.

A new, simple, sensitive and rapid spectrofluorimetric method has been developed for determination of certain adrenergic agonists such as isoxsuprine hydrochloride, ritodrine hydrochloride and etilefrine hydrochloride in their pure forms and pharmaceutical dosage forms. The method depends on micellar enhancement of the native fluorescence of investigated drugs by using 2% w/v sodium dodecyl sulfate (SDS) as an anionic surfactant. The enhanced fluorescence intensity of investigated drugs was measured at 305 nm after excitation at 278 nm. The interaction of studied drugs with SDS was studied, and the enhanced fluorescence intensity was exploited to develop an assay method for the determination of investigated drugs. The relative fluorescence intensity-concentration plots were rectilinear over the range 0.15-3.00 µg ml-1 , with low quantification limits of 0.132, 0.123 and 0.118 µg mL-1 for isoxsuprine, ritodrine and etilefrine, respectively. The proposed method was successfully applied for determination of studied drugs in their pharmaceutical formulations. Moreover, the high sensitivity of the proposed method allows performing the content uniformity testing of the studied drugs in their tablets by using the official United States Pharmacopeia (USP) guidelines. Statistical comparisons of the results with those of the reported methods revealed excellent agreement and indicated no significant difference in accuracy and precision.

Rapid analysis of ractopamine in pig tissues by dummy-template imprinted solid-phase extraction coupling with surface-enhanced Raman spectroscopy.

Ritodrine has similar skeleton structure to ractopamine and it was selected as the dummy-template molecule to synthesize the molecular imprinted polymers (MIPs). The MIPs exhibited better selectivity to ractopamine than to the dummy-template molecule: the imprint factor for ractopamine was 8.9, while 7.6 for ritodrine. The MIPs were used as sorbents in solid-phase extraction for selective enrichment of ractopamine, and some key parameters were optimized. After that, a rapid surface-enhanced Raman spectroscopy method was developed for analysis of ractopamine and isoxuprine in pig tissue samples. Under the optimal conditions, good linearity was achieved in the range of 20.0-200.0 µg/L for ractopamine and isoxsuprine at 842 cm(-1) and 993 cm(-1), respectively. The limits of detection were 3.1-4.3 µg/L, which were lower than the maximum allowed by U. S. Food and Drug Administration. The recoveries of ractopamine and isoxsuprine were 72.4-79.7% and 71.0-78.2% for the spiked pork and pig liver, respectively, while the relative standard deviations ranging from 7.4% to 13.0%. The results suggest that the proposed method is sensitive and selective, and it has good potential on the quantitative analysis of trace amounts of ß-agonists in complex samples.

Development and validation of fixed-time method for the determination of isoxsuprine hydrochloride in commercial dosages forms.

The main aim of this work was to develop a kinetic spectrophotometric method for the quantitative analysis of isoxsuprine hydrochloride in commercial tablets. The method is based on the reaction of isoxsuprine hydrochloride (ISx) with hydroxylamine hydrochloride and ammonium cerium (IV) nitrate in sulphuric acid medium at room temperature which resulted in the formation of yellow-coloured product peaking at 380 nm. The reaction is followed spectrophotometrically by measuring the absorbance as a function of time. Fixed time method (ΔA = A4-A2, where A2 and A4 refer to absorbance measurements taken at 2 and 4 min, respectively) was adopted for constructing the calibration curve which was found to be linear over the concentration range of 30-80 µgmL⁻¹ with molar absorptivity of 5.95 × 10³ L mol⁻¹ cm⁻¹. The method has been applied successfully to the determination of isoxsuprine hydrochloride in tablets. Statistical comparison (point and interval hypothesis tests) of the results showed that there is no significant difference between the proposed method and reference method.

Investigation of ractopamine molecularly imprinted stir bar sorptive extraction and its application for trace analysis of beta2-agonists in complex samples.

In this paper, a novel molecularly imprinted polymer (MIP) coated stir bar with ractopamine as template by glass capillary filling with magnetic core as substrate was prepared reproducibly. The ractopamine MIP coating was homogeneous and porous with the average thickness of 20.6 microm. The extraction apparatus for the stir bar was improved to avoid coating loss. The MIP-coated stir bar showed better extraction capacity and good selectivity than that of non-imprinted polymer (NIP) coated stir bar to ractopamine and its analogues. The extraction capacities of ractopamine, isoxsuprine, clenbuterol and fenoterol for MIP-coated stir bar were 3.3, 3.1, 2.8 and 2.4 times as much as that of the NIP coated stir bar, respectively. The MIP-coated stir bars could be used at least 40 times without apparent damage and kept in dried air for 8 months without reduce of extraction ability. A method for the determination of beta(2)-agonists in complex samples by MIP-coated stir bar sorptive extraction coupled with high-performance liquid chromatography (HPLC) was developed. The linear ranges were 0.5-40 microg/L for ractopamine and 1.0-40 microg/L for isoxsuprine and clenbuterol. The detection limits were within the range of 0.10-0.21 microg/L. The method was successfully applied to the analysis of beta(2)-agonists in spiked pork, liver and feed samples with the recoveries of 83.7-92.3%, 80.5-90.2% and 73.6-86.2%, respectively. The RSDs was within 2.9-8.1%. The method is very suitable for the determination of trace beta(2)-agonists in pork, liver and feed samples.

Simultaneous determination of ritodrine and isoxsuprine using coupling technique of synchronous fluorimetry and H-point standard addition method.

Simultaneous determination of two structurally related ss(2) adrenergic receptor agonists namely, Ritodrine HCl (RTH) and Isoxsuprine HCl (ISP) was performed using coupling technique of synchronous fluorimetry and H-point standard addition method. Under optimum conditions, linear determination ranges were 1.48 - 14.80 x 10(-6) mol L(-1) and 1.54 - 15.44 x 10(-6) mol L(-1) for ISP and RTH respectively. RTH and ISP could be determined simultaneously without interference from each other when their concentration ratio varies from 5:1 to 1:5 in the mixed sample. The proposed method was applied to the determination of RTH and ISP in synthetic mixture of pharmaceutical samples, the accuracy and precision of the results were satisfactory.

Determination of isoxsuprine hydrochloride by sequential injection visible spectrophotometry.

An automated sequential injection (SI) spectrophotometric method for the determination of isoxsuprine hydrochloride is described. The method is based on the condensation of aminoantipyrine with phenols (isoxsuprine hydrochloride) in the presence of an alkaline oxidizing agent (potassium hexacyanoferrate) to yield a pink colored product, the absorbance of which is monitored at 507 nm. Chemical as well as physical SI parameters that affect the signal response have been optimized in order to get better sensitivity, higher sampling rate and better reagent economy. Using the optimized aforesaid parameters, a linear relationship between the relative peak height and concentration was obtained in the range 1-60 mg l-1. The detection limit (as 3sigma value) was 0.3 mg l-1 and precision was 1.4% and 1.6% at 5 and 10 mg l-1, respectively. As compared to previous reports, wide linear range, low detection limit, and highly economical reagent consumption are the advantages of this automated method.

Determination of beta-agonist residues in bovine urine using liquid chromatography-tandem mass spectrometry.

A multiresidue method was developed and validated to screen bovine urine samples for 10 beta-2-adrenergic agonistic drugs--brombuterol, cimaterol, clenbuterol, clenpenterol, isoxsuprine, mabuterol, ractopamine, ritodrine, salbutamol, and tulobuterol--at the 2 microg/L level. The method is also quantitative in the range of 1 to 4 microg/L for all analytes except salbutamol. The procedure uses enzymatic digestion, liquid-liquid extraction, and cleanup on solid-phase extraction columns, followed by detection using a liquid chromatograph-tandem quadrupole mass spectrometer operated in the positive-ion atmospheric pressure chemical ionization multiple-reaction monitoring mode. Method validation included assessment of recoveries, repeatabilities, linearity of responses, decision limits, and detection capabilities. Overall average recoveries ranged from 70-91%; recoveries were generally lower for salbutamol. The decision limits ranged from 0.4-1.0 microg/L, and detection capabilities from 0.6-1.7 microg/L.

A GC-MS method for the determination of isoxsuprine in biological fluids of the horse utilizing electron impact ionization.

Isoxsuprine is used to treat navicular disease and other lower-limb problems in the horse. Isoxsuprine is regulated as a class 4 compound by the Association of Racing Commissioners, International (ARCI) and, thus, requires regulatory monitoring. A gas chromatography-mass spectrometry method utilizing electron impact ionization was developed and validated for the quantitation of isoxsuprine in equine plasma or equine urine. The method utilized robotic solid-phase extraction and tri-methyl silyl ether products of derivatization. Products were bis-trimethylsilyl (TMS) isoxsuprine and tris-TMS ritodrine, which released intense quantifier ions m/z 178 for isoxsuprine and m/z 236 for ritodrine that were products of C-C cleavage. To our knowledge, this procedure is faster and more sensitive than other methods in the literature. Concentrations in urine and plasma of isoxsuprine were determined from a calibrator curve that was generated along with unknowns. Ritodrine was used as an internal standard and was, therefore, present in all samples, standards, and blanks. Validation data was also collected. The limit of detection of isoxsuprine in plasma was determined to be 2 ng/mL, the limit of quantitation of isoxsuprine in plasma was determined to be < 5 ng/mL. The mean coefficient of determination for the calibrator curves for plasma was 0.9925 +/- 0.0052 and for calibrator curves for urine 0.9904 +/- 0.0075. The recovery efficiencies at concentrations of 50, 200, and 300 ng/mL were 76%, 73%, and 76%, respectively, in plasma and 92%, 89%, and 91% in urine.

Fluorimetric determination of isoxsuprine hydrochloride in pharmaceuticals and biological fluids.

Two simple and highly sensitive fluorimetric methods have been developed for the determination of isoxsuprine hydrochloride in bulk, in dosage forms and in biological fluids. The first method involves the direct measurement of the native fluorescence of the drug in the concentration range 0.4-4.0 microg ml(-1), the second method is based on the oxidation of isoxsuprine HCl with cerium(IV) followed by fluorimetric measurement in the concentration range 0.02-0.2 microg ml(-1). The average % found were 99.9 +/- 0.78 and 100.0 +/- 0.62 for the two methods, respectively. The minimum detectability (3 S(B)) were 0.11 and 0.007 microg ml(-1) for the two methods, respectively. The methods results showed insignificant difference with those of the official method.

Determination of phenolic sympathomimetic drugs in pharmaceutical samples and biological fluids by flow-injection chemiluminescence.

A rapid and highly sensitive flow-injection chemiluminometric method was developed for determination of 3 sympathomimetic drugs, namely, etilefrine hydrochloride, isoxsuprine hydrochloride, and prenalterol hydrochloride. The method is based on chemiluminescence induced by oxidation of drugs with acidified potassium permanganate in the presence of formic acid as a carrier. The calibration graphs were linear over the concentration ranges 0.2-9, 0.2-12.5, and 0.025-1.25 microg/mL for the 3 compounds, respectively. The method was applied successfully in determining the drugs in dosage forms and in biological fluids. A proposal for the reaction pathway is suggested.

Spectrophotometric determination of ritodrine and isoxsuprine hydrochlorides using 4-aminoantipyrine.

A simple, accurate, and rapid method for the quantitative determination of ritodrine hydrochloride (RTH) and isoxsuprine hydrochloride (ISH) in both pure and dosage forms, is described. The method is based on the development of pink colored product as a result of the condensation of 4-aminoantipyrine with phenols in the presence of an alkaline oxidizing agent. The resulting products are measured at 510 nm for both drugs, with molar absorptivities of 0.98 x 10(4) and 1.20 x 10(4) L/mol x cm for RTH and ISH, respectively. A study of the effect of commonly associated excipients revealed that they did not cause interference.

Use of ion-selective electrodes in kinetic flow injection: determination of phenolic and hydrazino drugs with 1-fluoro-2,4-dinitrobenzene using a fluoride-selective electrode.

A flow injection (FI) kinetic potentiometric method for the determination of phenolic (acetaminophen and isoxsuprine) and hydrazino (isoniazid) drugs is described. This work shows the usefulness of ion-selective electrodes as detectors in FI systems, not only for direct ion determination but also in routine kinetic analysis. The method is based on the reaction of 1-fluoro-2,4-dinitrobenzene (FDNB) with the analytes in a weakly alkaline medium, which proceeds through the liberation of fluoride from the reagent. The slow reactions with phenols are catalysed by micelles of cetyltrimethylammonium bromide. The reaction rate is monitored with a fluoride-selective electrode in a wall-jet configuration and is used to construct a calibration graph of antilog(delta E/S)-1 versus c (where E = potential, s = slope of the electrode and c = concentration), using the fixed-time approach. The response time and the long-term stability of the electrode were found to be adequate for such kinetic determinations. The proposed method overcomes problems associated with end-point spectrophotometric methods using FDNB and allows measurements in highly coloured or turbid solutions. The optimized method has a linear concentration range of 1 x 10(-4)-50 x 10(-4) mol dm-3, a measurement throughput of 20 or 40 per hour and the precision ranges from 1.8 to 3.6% relative standard deviation (n = 3). Results obtained for commercial pharmaceutical formulations compare favourably with those given by reference methods.

Automated flow injection spectrophotometric determination of para- and meta-substituted phenols of pharmaceutical interest based on their oxidative condensation with 1-nitroso-2-naphthol.

The reaction of para- and meta-substituted phenols with 1-nitroso-2-naphthol in the presence of either CeIV or PbIV as an oxidant has been used to develop a fast automated flow injection (Fl) method. A stopped-flow kinetic study of the reaction revealed the optimum conditions for the proposed Fl method. Acetaminophen, amoxicillin, cefadroxil, isoxsuprine, nylidrin, propylparaben, tyrosine and metaraminol can be determined in the range 1 x 10(-4)-8 x 10(-4) M, with relative standard deviations of less than 2%, and a measurement throughput of 40 measurements h-1. The method was evaluated by performing interference studies of common excipients and assaying commercial formulations of acetaminophen and isoxsuprine. The results were in good agreement with those obtained by acceptable spectrophotometric or high-performance liquid chromatographic methods (mean difference 2.1%). The high sample throughput of the Fl method was exploited by performing a content uniformity test of isoxsuprine tablets.

High-performance liquid chromatography of two peripheral vasodilators, nylidrin hydrochloride and isoxsuprine hydrochloride, in pharmaceutical dosage forms.

A reliable and selective high-pressure liquid chromatographic (HPLC) procedure for the quantitative determination of nylidrin hydrochloride or isoxsuprine hydrochloride in pharmaceutical dosage forms is described. The specificity of the stability-indicating HPLC procedure is presented for nylidrin hydrochloride.