Green Constant-wavelength Concurrent Selective Fluorescence Method for Assay of Ibuprofen and Chlorzoxazone in Presence of Chlorzoxazone Degradation Product.

Lower back pain is a universal dilemma leaving a negative effect on both health and life quality. It was found that a fixed dose combination of chlorzoxazone and ibuprofen gave a higher efficiency than analgesic alone in treatment of acute lower back pain. Based on the significant benefit of that combination, a green, sensitive, rapid, direct, and cost-effective method is created for concurrent determination of ibuprofen and chlorzoxazone in presence of 2-amino para chlorophenol (a synthetic precursor and potential impurity of chlorzoxazone) adopting the synchronous spectrofluorimetric technique. Synchronous spectrofluorimetric technique is adopted to avoid the highly overlapped native spectra of both drugs. The synchronous spectrofluorometric method was applied at Δλ = 50 nm, ibuprofen was measured at 227 nm while chlorzoxazone was measured at 282 nm with no hindering from one to another. The various experimental variables affecting the performance of the suggested technique were explored and adjusted. The suggested technique showed good linearity from 0.02 to 0.6 and 0.1 to 5.0 µg/mL for ibuprofen and chlorzoxazone, respectively. The produced detection limits were 0.27 × 10-3 and 0.03, while the quantitation limits were 0.82 × 10-3 and 0.09 µg/mL for ibuprofen and chlorzoxazone, respectively. The suggested approach was successfully applied for the analysis of the studied drugs in the synthetic mixture, different pharmaceutical preparations, and spiked human plasma. The suggested technique was validated with respect to the International Council of Harmonization (ICH) recommendations. The suggested technique was found to be simpler and greener with lower cost compared to the earlier reported methods which required complicated techniques, longer time of analysis, and less safe solvents and reagents. Green profile assessment for the developed method compared with the reported spectrofluorometric method was performed using four assessment tools. These tools confirmed that the recommended technique attained the most possible green parameters, so it could be used as a greener option in routine quality control for analyzing the two drugs in genuine form and pharmaceutical preparations.

Influence of solvent choice and operating conditions on Chlorzoxazone crystal shape and size

Abstract Solubility of pharmaceutical drugs in organic solvents is one of the important parameters to understand the equilibrium concentration of solute-solvent, which helps optimize and design crystallization conditions to obtain the desired product crystals. In the present study, Chlorzoxazone (CHZ) is used as a model pharmaceutical compound to investigate the equilibrium solubility, the influence of solvent and the operating conditions on the shape, and the size distribution. The solubility of CHZ is determined in organic solvents like Isopropanol, Ethanol, and 2-Ethoxyethylacetate, Ethylacetate and Ethyllactate using shake flask method from -5ºC to 60ºC. The solubility of CHZ in these solvents shows an increasing trend as the temperature increases in the following order: ethyllactate + water (0.5+0.5) < ethylacetate < isopropanol < ethanol < 2-ethoxyethylacetate < ethyllactate + water (0.75+0.25). The solvents, isopropanol, ethanol, and ethyl lactate, produce needle-shaped crystals, while 2-ethoxyethylacetate and ethyl acetate tend to produce plate shaped crystals. CHZ crystals obtained from 2-ethoxyethylacetate tend to have plate shaped crystals with a lower aspect ratio and are selected for batch cooling crystallization experiments performed at different cooling rates, and agitation. It is found that the agitation at 300 rpm and the cooling rate 0.2ºC/min produce more uniform crystal size distribution

Detection of adulterated drugs in traditional Chinese medicine and dietary supplements using hydrogen as a carrier gas.

Helium, a minor component of natural gas and radioactive minerals, is most commonly used as a carrier in gas chromatography-mass spectrometry (GC-MS). Its scarcity leads to limited availability and higher costs. In this experiment, hydrogen from a safe source of a hydrogen generator was tested as a substitutive carrier gas for the detection of adulterant in traditional Chinese medicine (TCM) and food supplements by GC-MS analysis. We found that the limits of detection (LODs) of using hydrogen were from 10 to 1000 µg/g. The levels of LODs tested among 170 drugs remain the same whether hydrogen or helium was used as a carrier gas with the exception of 7 drugs-benzbromarone, estradiol benzoate, bezafibrate, mefenamic acid, oxymetholone, piperidenafil and cetilistat. The real sample analysis results using hydrogen were as satisfactory as those using helium. In addition, the retention time was shortened after the chromatographic performance was optimized. In summary, it is worth considering hydrogen as a carrier gas due to its affordable costs, energy efficiency, carbon reduction and chromatographic advantages to detect adulterated drugs in TCM and dietary supplement using GC-MS.

Traditional versus advanced chemometric models for the impurity profiling of paracetamol and chlorzoxazone: Application to pure and pharmaceutical dosage forms.

Traditional Partial Least Squares (PLS) and Advanced Artificial Neural Network (ANN) models were applied for the quantitative determination of paracetamol (PAR) and chlorzoxazone (CZX) together with their process-related impurities namely; 4-aminophenol (AP), 4­chloroacetanilide (AC), 4­nitrophenol (NP), 4­chlorophenol (CP) and 2­amino-4-chlorophenol (ACP). Both models were applied first to full spectrum data then the results were compared to those obtained after wavelength selection using Genetic Algorithm (GA). A 5-level 7-factor experimental design was used giving rise to 25 mixtures containing different proportions of the seven compounds. The calibration set was composed of 15 mixtures while 9 mixtures were used in the validation set to test the predictive ability of the suggested models. The four models PLS, ANN, GA-PLS and GA-ANN were successfully applied for the determination of PAR and CZX in their pure and pharmaceutical dosage form. One way ANOVA was carried out between the developed methods and the official ones for PAR and CZX and no significant difference was found. The four models can be easily applied for the determination of the selected drugs in quality control laboratories lacking expensive HPLC instruments.

Simultaneous Determination of Paracetamol and Chlorzoxazone in Their Combined Pharmaceutical Formulations by Reversed-phase Capillary Liquid Chromatography Using a Polymethacrylate Monolithic Column.

Recently, analytical separation techniques have the potential toward green approaches to reduce the environmental impact. This study focuses on the development of an analytical method for the determination of paracetamol and chlorzoxazone in their pharmaceutical combination. The separation was achieved using a home-made capillary column (0.10 mm i.d. × 200 mm length) filled with porous cross-linked hexyl polymethacrylate as monolithic stationary phase. The method proved to be simple, fast, sensitive, efficient, cost-effective and green approach due to the combination of the amazing properties of a monolithic material and a miniaturized liquid chromatography, which would be considered as a step toward reducing the analytical costs and the environmental impact of chromatographic applications. Both components were detected using a 3-nL nano-UV cell fixed at 270 nm wavelength. The optimized mobile phase was composed of 1% aqueous formic acid solution and acetonitrile at 40:60 ratio, 1.0 µL/min flow rate, 4.0 nL injection volume and 50°C column temperature. Under the optimized conditions, paracetamol and chlorzoxazone have been separated in about 6.5 min with chromatographic resolution of 2.37. The prepared column and the analysis method was fully validated and compared with other reported works. All findings allow to conclude that the prepared column and proposed method are applicable for quality control and routine analysis of the two drugs.

Novel pure component contribution, mean centering of ratio spectra and factor based algorithms for simultaneous resolution and quantification of overlapped spectral signals: An application to recently co-formulated tablets of chlorzoxazone, aceclofenac and paracetamol.

In this work, resolution and quantitation of spectral signals are achieved by several univariate and multivariate techniques. The novel pure component contribution algorithm (PCCA) along with mean centering of ratio spectra (MCR) and the factor based partial least squares (PLS) algorithms were developed for simultaneous determination of chlorzoxazone (CXZ), aceclofenac (ACF) and paracetamol (PAR) in their pure form and recently co-formulated tablets. The PCCA method allows the determination of each drug at its λmax. While, the mean centered values at 230, 302 and 253nm, were used for quantification of CXZ, ACF and PAR, respectively, by MCR method. Partial least-squares (PLS) algorithm was applied as a multivariate calibration method. The three methods were successfully applied for determination of CXZ, ACF and PAR in pure form and tablets. Good linear relationships were obtained in the ranges of 2-50, 2-40 and 2-30µgmL(-1) for CXZ, ACF and PAR, in order, by both PCCA and MCR, while the PLS model was built for the three compounds each in the range of 2-10µgmL(-1). The results obtained from the proposed methods were statistically compared with a reported one. PCCA and MCR methods were validated according to ICH guidelines, while PLS method was validated by both cross validation and an independent data set. They are found suitable for the determination of the studied drugs in bulk powder and tablets.

A novel organic-inorganic hybrid monolithic column prepared in-situ in a microchip and its application for the determination of 2-amino-4-chlorophenol in chlorzoxazone tablets.

γ-Alumina nanoparticles (γ-Al2O3) were introduced to the conventional poly(methacrylic acid-co-ethylene glycol dimethacrylate) (MAA-co-EGDMA) monolith to prepare a novel organic-inorganic hybrid monolith, poly(MAA-co-EGDMA)-Al2O3 monolith. The polymerization was induced in-situ with UV irradiation in an ultraviolet transparent polymethyl methacrylate (PMMA) microfluidic chip. The monolith-based solid phase microextraction system was used for the on-line determination of 2-amino-4-chlorophenol (ACP) in chlorzoxazone tablets coupled with an optical fiber spectrophotometer. Several parameters affecting the adsorption/desorption, including sample pH value, sample flow rate, sampling time, eluent flow rate, and eluting time, were investigated in detail. Under the optimized conditions, limit of detection (LOD) and limit of quantification (LOQ) of the method were calculated to be 2.8 and 9.1 µg L(-1), respectively, with the relative standard deviation (RSD) of 3.1%.

Multivariate optimization of separation conditions for simultaneous determination of acemetacin and chlorzoxazone in a pharmaceutical preparation by HPLC using response surface methodology.

A new, fast, accurate, precise, and sensitive RP-HPLC method for the simultaneous determination of acemetacin and chlorzoxazone has been developed. Response surface methodology with a central composite design was used to optimize the acetonitrile and ammonium acetate percentage in the mobile phase and pH of ammonium acetate. The optimum separation was achieved on a C18 column (250 x 4.6 mm id, 5 microm particle size) using the mobile phase methanol-acetonitrile-0.02 M ammonium acetate, pH 9.4 (25 + 35 + 40, v/v/v) at a flow rate of 1.5 mL/min; UV detection at 270 nm, and cyanocobalamin as an internal standard. This developed method was validated and successfully applied to a coated tablet pharmaceutical preparation.

Development and validation of a simple LC method for the determination of phenacetin, coumarin, tolbutamide, chlorzoxazone, testosterone and their metabolites as markers of cytochromes 1A2, 2A6, 2C11, 2E1 and 3A2 in rat microsomal medium.

Cytochrome P450 enzymes are responsible for the oxidative metabolism of most pharmaceutical compounds. A "cocktail" approach which employs simultaneous administration of a mixture of substrates of CYP enzymes was often used to assess the metabolic activity of multiple P450 forms in one experiment. Phenacetin, coumarin, tolbutamide, chlorzoxazone and testosterone are commonly used as probe substrates to evaluate cytochrome P450 function. An analytical strategy to simultaneously extract and analyze the five probe substrates and their major metabolites by HPLC-DAD was developed. The incubation was done with all the substrates in one step. The ten analytes were extracted simultaneously by solid-phase extraction (SPE) from rat liver microsomes. A C18 analytical column and mobile phase composed of acetonitrile and 0.02% aqueous phosphoric acid were used for the chromatographic separation with DAD detection. Limits of quantification varied between 0.02378 and 0.2361 microg/mL which contributed to quantify all these drugs and metabolites with UV detection. The method is applicable for the modeling and description of pharmacological interactions on rat cytochromes P450 or can be used for in vitro evaluation of cytochromes 1A2, 2A6, 2C11, 2E1 and 3A2.

Stability-indicating TLC-densitometric method for simultaneous determination of paracetamol and chlorzoxazone and their toxic impurities.

A highly sensitive, selective and accurate thin-layer chromatographic (TLC)-densitometric method has been developed and validated for the simultaneous determination of paracetamol (PAR) and chlorzoxazone (CZ) and their toxic impurities, 4-amino phenol (4AP) and 2-amino-4-chlorophenol (2ACP), respectively, which are also considered to be the hydrolytic degradation products and related substances of the studied drugs. A developing system consisting of chloroform-methanol-glacial acetic acid (9.5:0.5:0.25, by volume) was found to be sufficient for chromatographic separation among the four studied components using pre-activated silica gel 60 F254 TLC plates with ultraviolet detection at 225 nm. Calibration curves were constructed in the ranges of 0.3-3, 1-10, 0.06-3 and 0.04-3 µg/band for PAR, CZ, 4AP and 2ACP, respectively, using polynomial equations. The developed method was validated according to International Conference on Harmonization guidelines and demonstrated good accuracy and precision. Moreover, the method was successfully applied for the determination of PAR and CZ in different marketed samples and the results were statistically compared to those obtained by the reported reversed-phase high-performance liquid chromatography method using F-test and Student's-t test. The low detection and quantitation limits of the developed method make it suitable for quality control and stability studies of PAR and CZ in different pharmaceutical formulations.

Development and validation of packed column supercritical fluid chromatographic technique for quantification of chlorzoxazone, paracetamol and aceclofenac in their individual and combined dosage forms.

A reproducible, rapid and sensitive method has been developed for the assay of chlorzoxazone (CHL), paracetamol (PCM) and aceclofenac (ACE) in their combined solid dosage forms using packed-column supercritical fluid chromatography (SFC). The analytes were resolved by elution with supercritical carbon dioxide doped with 15% v/v methanol as the modifier on an ACE 5 Phenyl column (150 × 4.6 mm, 5 µm). The detection was carried out at 215 nm using a UV-Visible detector. The densities and polarities of the mobile phase were optimized from the effects of pressure, temperature and modifier concentration on chromatographic parameters like retention time, retention factor, resolution, asymmetry and theoretical plates. Modifier concentration proved to be the most effective means for changing both retention and selectivity. The developed method was validated as per International Conference on Harmonization guidelines. The developed SFC method was compared with a reported high-performance liquid chromatography method for the estimation of CHL, PCM and ACE using Student t-test. With respect to the speed and use of organic solvents, SFC was found to be superior and eco-friendly. The developed SFC method was successfully used for the assay of different marketed formulations containing CHL, PCM and ACE individually and in combination.

Spectrofluorimetric assessment of chlorzoxazone and ibuprofen in pharmaceutical formulations by using Eu-tetracycline HCl optical sensor doped in sol-gel matrix.

A novel, simple, sensitive and selective spectrofluorimetric method was developed for the determination of trace amounts of chlorzoxazone and Ibuprofen in pharmaceutical tablets using optical sensor Eu-Tetracycline HCl doped in sol-gel matrix. The chlorzoxazone or Ibuprofen can remarkably enhance the luminescence intensity of Eu-Tetracycline HCl complex doped in a sol-gel matrix in dimethylformamide (DMF) at pH 9.7 and 6.3, respectively, λ(ex) = 400 nm. The enhancing of luminescence intensity peak of Eu-Tetracycline HCl complex at 617 nm is proportional to the concentration of chlorzoxazone or Ibuprofen a result that suggested profitable application as a simple optical sensor for chlorzoxazone or Ibuprofen assessment. The dynamic ranges found for the determination of chlorzoxazone and Ibuprofen concentration are 5 × 10(-9)-1 × 10(-4) and 1 × 10(-8)-7 × 10(-5) mol L(-1), and the limit of detection (LOD) and quantitation limit of detection (LOQ) are 3.1 × 10(-10), 9.6 × 10(-10) and 5.6 × 10(-10), 1.7 × 10(-9) mol L(-1), respectively.

Determination of brain cytochrome P450 2E1 activity in rat with the probe of chlorzoxazone by liquid chromatography-mass spectrometry.

A simple and sensitive method was developed for the determination of cytochrome P450 2E1 (CYP2E1) activity based on the liquid chromatography-mass spectrometry (LC-MS) analysis of 6-hydroxychlorzoxazone generated by 6-hydroxylation of chlorzoxazone under specific catalysis of CYP2E1. In the proposed method, 2-benzoxazolinone was chosen as internal standard and isopropyl ether was used as extraction solvent for sample preparation. The inter-day and intra-day precisions at low, medium and high concentrations of 6-hydroxychlorzoxazone were below 20.0%, and the LOD (S/N=3) was 0.05 ng/mL. This method was applied to analyze the CYP2E1 activity of rat in different brain regions including frontal cortex (FC), cerebellum (CB), brain stem (BS), hippocampus (HC), striatum (ST), thalamus (TH), and olfactory bulb (OB). The results confirmed that chlorzoxazone was a suitable probe for the determination of CYP2E1 activity in brain regions and samples with low content of CYP2E1.

Simultaneous determination of aceclofenac, paracetamol, and chlorzoxazone by RP-HPLC in pharmaceutical dosage form.

A simple, rapid, and precise reversed-phase liquid chromatographic method is developed for simultaneous determination of paracetamol, aceclofenac, and chlorzoxazone in their ternary mixtures of commercial pharmaceutical preparation. This method uses a Zorbax SB C18, 250 x 4.6 mm, 5 microm analytical column. Mobile phase is acetonitrile and buffer (40:60, v/v), buffer containing 50 mM orthophosporic acid; pH of the buffer is adjusted to 6 with 10% w/v sodium hydroxide solution. The instrumental settings are at a flow rate of 1 mL/min; the column temperature is 25 degrees C, and detector wavelength is 270 nm. The sample concentrations are measured on weight basis to avoid the internal standard. The method is validated and shown to be linear. The correlation coefficients for paracetamol, aceclofenac, and chlorzoxazone are 0.9981, 0.9990, and 0.9986, respectively. The recovery values for paracetamol, aceclofenac, and chlorzoxazone ranged from 100.7-101.4%, 100.4-101.0%, and 100.5-101.3%, respectively. The relative standard deviation for six replicates is always less than 2%. This HPLC method is successfully applied to the simultaneous quantitative analysis of the title drugs in tablets and can be applied for assay and dissolution test of tablets for the estimation of paracetamol, aceclofenac, and chlorozoxazone in their commercial samples.

Simultaneous determination of chlorzoxazone and ketoprofen in binary mixtures and in ternary mixtures containing the chlorzoxazone degradation product by reversed-phase liquid chromatography.

New, simple, rapid, and precise reversed-phase high-performance liquid chromatographic (LC) methods were developed for the simultaneous determination of chlorzoxazone (CH) and ketoprofen (KT) in binary mixtures and in ternary mixtures containing the CH degradation product, 2-amino-4-chlorophenol (CD). The analytes were separated by LC on a Lichrosphere 60 C18 column (250 x 4 mm, 5 microm). The mobile phases, methanol-water (40:60, v/v) at 1 mL/min and methanol-0.05% phosphoric acid (60:40, v/v, pH 2.81) at 1.5 mL/min, satisfactorily resolved the binary and ternary mixtures, respectively. The UV detector was operated at 280 nm for the determination of CH and at 254 nm for the determination of KT and CD. Linearity, accuracy, and precision were found to be acceptable over the concentration ranges of 20-240 and 5-60 microg/mL for CH and KT, respectively, in the binary mixtures and 50-300, 10-60, and 20-160 microg/mL for CH, KT, and CD, respectively, in the ternary mixtures. The optimized methods proved to be specific, robust, and accurate for the quality control of CH and KT in pharmaceutical preparations.

Simultaneous determination of paracetamol, chlorzoxazone, and related impurities 4-aminophenol, 4'-chloroacetanilide, and p-chlorophenol in pharmaceutical preparations by high-performance liquid chromatography.

This paper presents a simple, specific, and precise high-performance liquid chromatographic method for the simultaneous determination of paracetamol (PCM), chlorzoxazone (CXZ), and their related impurities in bulk raw materials and solid dosage forms. The mobile phase consisted of water-methanol-glacial acetic acid (60 + 40 + 2, v/v/v). A column containing octadecylsilane chemically bonded to porous silica particles (Spherisorb ODS 1, 25 cm x 4.6 mm, 5 microm) was used as stationary phase. Detection was performed using a variable wavelength ultraviolet-visible detector set at 272 nm for all compounds. Solutions were injected into the chromatograph under isocratic condition at a constant flow rate of 1.2 mL/min. The method was validated according to International Conference on Harmonization requirements and demonstrates good accuracy and precision and a wide linearity range. The method separates PCM, CXZ, and 3 major impurities [4-aminophenol (4AP), 4'-chloracetanilide (4CA), and p-chlorophenol (PCP)] with fair resolution in less than 15 min. The developed method is rapid and sensitive (limit of detection for 4AP, 4CA, and PCP established at 31.25, 39.06, and 65.16 ng/mL, respectively) and, therefore, suitable for quality control and stability studies of these compounds in dosage forms.

[Ratio spectrophotometry and its application to assay of compound pharmacy preparations].

The present paper is to study ratio spectrophotometry for its application to the assay of compound pharmacy preparations. According to the feature of ratio spectra, the wavelength pair was selected at the peak point or valley point of ratio spectra to establish ratio spectrophotometry for the determination of Co-Chlorzoxazone tablet and injection of caffeine and sodium benzoate. The average recoveries and relative standard deviations for Chlorzoxazone and paracetamol were 100.0% and 1.28%, and 100.0% and 0.84%, respectively; the average recoveries and relative standard deviations for caffeine and sodium benzoate were 99.98% and 0.70%, and 99.91% and 0.78%, respectively. It is concluded that as long as the ratio spectra exhibit at least one peak and one valley, the results are satisfactory.

Robustness of chlorzoxazone as an in vivo measure of cytochrome P450 2E1 activity.

AIMS: Chlorzoxazone is metabolized by cytochrome P450 2E1 (CYP2E1) to a single oxidized metabolite, 6-hydroxychlorzoxazone. The aim of the study was to test the robustness of chlorzoxazone as an in vivo probe of CYP2E1 activity in humans, with emphasis on investigating short-term and long-term intra-individual variabilities and effects of different doses of the drug. In addition, the influences of body build, drug metabolizing enzyme genotype, blood sampling time, and moderate recent ethanol intake were investigated. METHODS: The 6-hydroxychlorzoxazone:chlorzoxazone (metabolic) ratio in plasma was measured at 2 h in 28 male and nine female volunteers following a single oral dose of 500 mg chlorzoxazone. Similarly, the metabolic ratios at 4 h and 6 h were measured in 20 of the males. The metabolic ratio at 2 h was also determined 1.5 and 2.5 years later in 13 and seven males, respectively, and weekly for 3 weeks in seven males, after a dose of 500 mg, once at higher (750 mg) and lower (250 mg) doses, and once (500 mg) following moderate ethanol intake (0.5 g kg(-1) body weight) the preceding evening. Genotypes were determined for CYP2E1 as well as for N-acetyltransferase 2 and glutathione transferase M1. RESULTS: Excluding an outlier (ratio = 1.6) the metabolic ratio at 2 h ranged from 0.12 to 0.61 (n = 36). A positive correlation with body weight (r = 0.61, P < 0.001) suggested dose-dependent metabolism of chlorzoxazone. The metabolic ratio decreased with increasing chlorzoxazone dose (P = 0.01), again suggesting dose-dependent metabolism. Long-term (yearly intervals) and short-term (weekly intervals) intra- and interindividual variabilities in metabolic ratio were similar (30% and 63%vs 28% and 54%, respectively). Both inter- and intra-individual variabilities tended to decrease with increasing dose of chlorzoxazone. There was no significant influence of moderate ethanol intake the preceding evening, or of CYP2E1 genotype on the metabolic ratio. CONCLUSIONS: The relatively low intra-individual variability in the metabolism of chlorzoxazone suggests that a single-sample procedure may suffice to assess CYP2E1 activity in vivo. However, chlorzoxazone metabolism is dose-dependent at commonly used doses and it is therefore advisable to adjust the dose for body weight. Moderate intake of ethanol the preceding evening did not significantly affect the chlorzoxazone metabolic ratio.

Simultaneous determination of aspirin and isosorbide 5-mononitrate in formulation by reversed phase high pressure liquid chromatography.

A simple, precise and rapid reversed phase HPLC method was developed for the simultaneous estimation of aspirin (AS) and isosorbide 5-mononitrate (ISM) in combined formulation. The method was carried out on a Thermo Quest C18 column using a mixture of water:methanol (water pH adjusted to 3.4 using dilute orthophosphoric acid) and detection was carried out at 215 nm using chlorzoxazone as internal standard. Both the drugs showed linearity in the range of 2-10 microg/ml and limits of quantification was found to be 4 and 40 ng/ml for AS and ISM, respectively.

Determination of 6-hydroxychlorzoxazone and chlorzoxazone in porcine microsome samples.

A simple, accurate and sensitive HPLC method for the in vitro determination of 6-hydroxychlorzoxazone and chlorzoxazone in porcine microsome samples is described. Chromatography was performed on a YMC-Pack ODS-AQ column using a mobile phase of 0.05% phosphoric acid pH 3-methanol (60:40, v/v). UV detection was carried out at 287 nm. The detector response was linear over the concentration range 25-2000 ng/ml. This assay produced quick, accurate, and repeatable results.