A stability-indicating HPLC method for the determination of glucosamine in pharmaceutical formulations

A stability-indicating high performance liquid chromatographic (HPLC) method was developed for the assay of glucosamine in bulk forms and solid dosage formulations. The HPLC separation was achieved on a Phenomenex Luna amino column (150 mm x 4.6 mm, i.d., 5 microm particle size) using a mobile phase of acetonitrile-phosphate buffer (75:25, v/v, pH 7.50) at a flow rate of 1.5 ml min(-1) and UV detection at 195 nm. The method was validated for specificity, linearity, solution stability, accuracy, precision, limit of detection, and limit of quantitation. The detector response for glucosamine hydrochloride was linear over the selected concentration range from 1.88 to 5.62 mg ml(-1) with a correlation coefficient 0.9998. The accuracy was between 98.9 and 100.5%. The precision (R.S.D.) amongst six sample preparations was 1.1%. The limit of detection and the limit of quantitation are 0.037 and 0.149 mg ml(-1), respectively. The sample and standard solutions were stable for 1 week. The method was successfully used for analysis of active-excipient compatibility samples used for development of a solid dosage formulation in our laboratory and subsequent stability studies. The method was also used for the analysis of glucosamine in several commercially available solid dosage forms.     

Development of HPLC method for the determination of levosulpiride in human plasma

A rapid and simple high performance liquid chromatography (HPLC) method was developed and validated for determination of levosulpiride in human plasma. After extraction with ethylacetate/methylene chloride (5:1, v/v), analysis of levosulpiride in plasma samples was carried out using a reverse phase C18 column with fluorescence detector (maximum excitation at 300 nm and maximum emission at 365 nm) for separation and quantification. A mixture of methanol-20 mM phosphate buffer (pH 3.5, 16:84, v/v) was used as a mobile phase. The method was specific and sensitive with a limit of quantification of 5 ng/ml. This HPLC method was validated by examining the precision and accuracy for inter- and intra-day analysis in the concentration range of 5-150 ng/ml. The relative standard deviation (R.S.D.) in inter- and intra-day validation were 8.16-19.75 and 3.90-11.69%, respectively. In stability tests, levosulpiride in human plasma was stable during the storage and assay procedure. The method was applied to the bioequivalence study of two levosulpiride tablet formulations (25 mg) after a single oral administration.     

An high performance liquid chromatographic method for the quantification of cotinine in the urine of preschool children

Tobacco smoke exposure is an important and preventable cause of morbidity among children. Enviromental tobacco smoke (ETS) increases respiratory symptoms and disease and also decreases lung function in children who live in a household with at least one smoker. We have developed a simple and reliable HPLC method with diode array dedection to determine the urine concentrations of cotinine in children aged 3 to 6 years, exposed to ETS. The assay involved a liquid-liquid extraction with chloroform. The HPLC method utilized a Chromasil C18 column (150 mm x 4.6 mm i.d.) and an isocratic mobile phase of phosphate buffer: acetonitrile (83:17 v/v, 0.02 M containing 0.1% triethylamine, adjusted to pH 6.72 with orthophosphoric acid), at a flow rate of 0.7 ml min(-1). The detection was performed at 260 nm and the total analysis time of analysis was less than 15 min. Linearity ranged from 0 to 80 microg L(-1); correlation coefficients (r2) for calibration curves were greater than 0.99. With 2 mL of urine for extraction, the limit of detection was 0.1 microg L(-1). The mean extraction ratio of cotinine was 88.78%. This analytical method is suitable for the determination of cotinine levels in a large number of urine samples.     

LC determination of the anti-ulcer agent CDRI-85/92 in rat serum

CDRI-85/92 is a new potent anti-ulcer compound developed by Central Drug Research Institute, Lucknow (India). This compound is in advanced stage of preclinical trials. A High-performance liquid chromatographic (HPLC) method was developed and validated for the analysis of CDRI-85/92 using rat serum. The HPLC analysis, applicable to 0.5-ml volumes of serum, involved protein precipitation of serum samples with acetonitrile (1:3 v/v) followed by centrifugation and separation on a C-18 column and the use of UV detector at the wavelength 250 nm. The method was sensitive with a lowest limit of quantitation (LLOQ) of 1.25 ng ml⁻¹ in rat serum and the recovery was more than 96%. The linearity was satisfactory as indicated by correlation of >0.99, in addition to the visual examination of the calibration curves. The precision and accuracy were acceptable as indicated by relative standard deviation (R.S.D.) ranging from 4.15 to 8.21%, bias values ranging from 2.96 to 11.18%. In-process stability evaluation showed the stability of the compound in processed samples lasted up to 168 h. The method was applied for analysing CDRI-85/92 in rat serum after administration of single oral or iv bolus dose of 20 mg kg⁻¹. The robustness/ruggedness of the HPLC procedure was tested using different HPLC instrumentation and column of different make. The assay was found to be sensitive (limit of quantification was 1.25 ng ml⁻¹), specific (retention time for CDRI-85/92 is 7.5 min), accurate (% bias is <12%), precise (% R.S.D. is <10%), robust (no significant change in peak profile in two HPLC Instruments) and reliable for use in pharmacokinetic or toxicokinetic studies.     

Sensitive LC determination of ciprofloxacin in pharmaceutical preparations and biological fluids with fluorescence detection

A simple and highly sensitive isocratic reversed-phase high-performance liquid chromatographic method (RP-HPLC) has been developed for the determination of ciprofloxacin. Separation of ciprofloxacin and anthranilic acid (internal standard) was achieved on a Kromasil 100, C(18), 5 microm (250 x 4.6 mm i.d.) reversed-phase column, using fluorescence detection with lambda(exc)=300 nm and lambda(emi)=458 nm. The mobile phase consisted of acetonitrile-methanol-acetate buffer (pH 3.60; 0.05 M) (10:30:60 v/v/v) containing 1% v/v acetic acid. The analysis was performed in less than 9 min, with a flow rate of 0.8 ml min(-1). A rectilinear relationship was observed for concentrations between 0.005 and 1.0 microg ml(-1) of ciprofloxacin in aqueous standard solutions and serum and the detection limit was 20 pg injected on-column. The intra- and inter-day relative standard deviation (n=8) ranged from 1.6 to 2.6% and from 1.9 to 4.8%, respectively, calculated at three concentration levels of standard solutions. Direct measurements of ciprofloxacin in pharmaceutical preparations and in serum, after precipitation of proteins, were performed with high precision and accuracy. The application of the method to urine samples involved a solid-phase extraction treatment of the samples using C(18) cartridges. The linear working range in urine extended from 0.05 to 2.0 microg ml(-1) and the detection limit was 0.2 ng injected on-column.     

A detection scheme for paraquat poisoning: validation and a five-year experience in Australia

We report the validation of a quantitative method for paraquat in plasma and urine using high-performance liquid chromatography (HPLC) with ultraviolet detection (260 nm). Furthermore, we illustrate the use of this method in the clinic (over five years), in conjunction with a qualitative urine paraquat screen. Urine or plasma sample (1 mL) preparation was performed in duplicate using C18 solid-phase extraction. Chromatographic separation was achieved on a Zorbax RX-Silica column (250 x 4.6-mm i.d.). The mobile phase consisted of 96% sodium chloride (5 g/L) and 4% acetonitrile (pH 2.2) pumped at 1.0 mL/min. Using a single-point calibration (1.0 mg/L), the method was found to be linear from 0.1 to 5.0 mg/L. The accuracy and imprecision of the method, over the linear range and for plasma and urine, were 94.7-104.9% and < 12.2%, respectively. The limit of quantitation for both matrices was 0.1 mg/L. The absolute recovery of paraquat from plasma and urine was 79.9 +/- 5.3% and 88.2 +/- 5.3%, respectively. From January 1995 to February 2000, 47 qualitative urine paraquat screens were requested throughout Australia. Nine screens were positive, and eight were confirmed to have paraquat present by our HPLC method. One sample was not analyzed by HPLC because the patient died prior to analysis. Thus, no false-positive results were reported for the qualitative urine screen. An additional 11 samples were referred for patients with positive screens from other sites for HPLC confirmation. The presence of paraquat was confirmed in nine of these samples. In conclusion, a qualitative urine screen combined with our validated HPLC confirmation is an effective protocol for assessing suspected cases of paraquat poisoning.     

Drug monitoring and toxicology: a simple procedure for the monitoring of felbamate by HPLC-UV detection

This article describes a simple isocratic high-performance liquid chromatographic (HPLC) method with UV detection for the determination of felbamate in the serum of patients with epilepsy. Sample preparation requires only protein precipitation with a single-step methanol extraction. After centrifugation, the resulting supernatant was injected directly onto the HPLC system. Separation was achieved by reversed-phase HPLC, using a 5-microm Microsorb-MV C(18) column (250 x 4.6 mm) connected to a Silica C(18) guard column (20 x 4.6 mm) and a mobile phase consisting of a mixture of phosphate buffer (pH = 6.9, 0.05 M), methanol, and acetonitrile (64:18:18, v/v/v). The flow rate was at 1.0 mL/min and column temperature was set at 35 degrees C. Quantitation was performed by measurement of the UV absorbance at a wavelength of 210 nm. Calibration curves were linear over a range of 2-200 mg/L, which covered the proposed range of 50-150 mg/L for reference. Both within-run and between-run precision were lower than 5%. Recoveries ranged between 97% and 105% for spiked and pooled samples. No interferences with other common antiepileptic drugs (except zonisamide) were observed. The method requires only 100 microL of serum or less. It is simple and fast (sample preparation and analysis time) and suitable for routine clinical use.     

Enantioselective determination of cetirizine in human urine by HPLC

In order to study the simultaneous determination of (+)- and (-)-cetirizine in human urine we have developed a chiral separation method by HPLC. A chiral stationary phase of alpha1-acidglycoprotein, the AGP-CSP, was used to separate the enantiomers. The pH of the phosphate buffer, as well as the content of the organic modifier in the mobile phase, markedly affected the chromatographic separation of (+)- and (-)-cetirizine. A mobile phase of 10 mmol/l phosphate buffer (pH 7.0)-acetonitrile (95: 5, v/v) was used for the urine assays. Ultraviolet absorption was monitored at 230 nm and roxatidine was employed as the internal standard for quantification. (+)-Cetirizine, (-)-cetirizine and the internal standard were eluted at retention times of 12, 16, and 32 mins, respectively. The detection limit for cetirizine enantiomers was 400 ng/ml of urine. A pharmacokinetic study was conducted with the help of 5 healthy female volunteers who were administered with a single oral dose of racemic cetirizine (20 mg). The peak area ratios provided by the cetirizine enantiomers were linear (r>0.997) over a concentration range of 2.5-200 microg/ml. The peak of the excreted cetirizine enantiomers appeared in the urine sample during the period of 1-2 hrs following the administration of the oral dose. The excreted level of (+)-cetirizine was slightly higher than (-)-cetirizine but the difference was not statistically significant. However, this method appears to have applications for enantioselective pharmacokinetic studies of racemic drugs.     

Analysis of anticancer drugs in biological fluids: determination of taxol with application to clinical pharmacokinetics

Taxol, a novel antimitotic, antitumor agent is currently undergoing Phase 1 clinical trials for the treatment of various tumors. An isocratic HPLC method has been developed for the determination of taxol in human plasma and urine. The method was then applied to the clinical pharmacokinetics of taxol following 6-h intravenous (i.v.) infusions at doses of 175 and 225 mg m-2. A mobile phase of methanol-acetate buffer (0.02 M, pH 4.5) (65:35, v/v) was used to elute a C8 column with detection at 227 nm. The sample preparation involved extraction with t-butyl methyl ether followed by further clean-up of the sample by solid-phase extraction. The method was linear from 0.10-10 microM injected, with a chromatographic run time of 6 min. The results obtained from the clinical study indicate that the plasma pharmacokinetics of taxol are best characterized by a two compartment open body model. Additionally, the present study resulted in the detection of a previously unreported peak which may be a metabolite of taxol.     

HPLC-UV method development and validation for 16-dehydropregnenolone, a novel oral hypolipidaemic agent, in rat biological matrices for application to pharmacokinetic studies

An accurate and precise HPLC assay has been developed and validated for determination of dehydropregnenolone (DHP) in rat plasma, bile, urine and feces. Separation was achieved using a C-18 reversed phase column with a mobile phase comprising of acetonitrile and deionized water (55:45% v/v) using a UV detector, set at a wavelength of 248 nm. The method, applicable to 200-microl plasma, bile and urine, involved double extraction of the samples with n-hexane. The sample clean up for feces involved single extraction of 50 mg of sample with 3 ml of acetonitrile. The method was sensitive with a limit of quantitation of 20 ng/ml in all the matrices and absolute recovery >92%. Precision and accuracy were within the acceptable limits, as indicated by relative standard deviation varying from 4.7 to 11.2% and bias values ranging from 1.8 to 8.8%. Moreover, DHP was stable in plasma, bile and urine up to 90 days of storage at -60 degrees C and after being subjected to three freeze-thaw cycles. The method was applied to generate the pharmacokinetics of DHP in rats after oral and intravenous administration.     

Evaluation of the EMIT Mycophenolic Acid Assay from Dade Behring

The Emit Mycophenolic Acid Assay, a new homogeneous enzyme immunoassay for the quantitative analysis of mycophenolic acid (MPA) in human plasma, was evaluated and compared to a high-performance liquid chromatography (HPLC) method. Coefficients of variation (CV) of the within-run imprecision (n = 10) varied from 2.5% to 4.4% and from 1.3% to 4.9% for the Emit and the HPLC, respectively. The CV's of between-day imprecision (n = 10) ranged from 7.9% to 10.8% for the Emit and from 4.7% to 12.1% for the HPLC. Mean recoveries were 95.6% and 100.1% for Emit and HPLC, respectively. Serial dilution of a patient pool demonstrated a linear relationship between expected (x) and measured (y) concentrations: Emit, y = 0.998x + 0.086; HPLC, y = 1.006x - 0.016. The detection limit and the lower limit of quantification were 0.087 mg/L and 0.20 mg/L for Emit. The detection limit for HPLC was 0.08 mg/L using a signal-to-noise ratio of three. Sample stability under various storage conditions was satisfactory, although storage at -20 degrees C is recommended for storage longer than one day. No cross-reactivity from the major metabolite mycophenolic acid glucuronide (MPAG) was found. A correlation study on 261 patient samples yielded the following regression equation (bivariate Deming procedure): Emit = 1.012HPLC + 0.244, r = 0.970.     

Direct measurement of salicylphenolic glucuronide in human urine

Indirect measurement of salicylphenolic glucuronide (SPG) has suggested that the formation of this metabolite from therapeutic doses of salicyclic acid (SA) is capacity-limited in humans. A direct high performance liquid chromatographic (HPLC) assay for SPG in human urine is described. SPG was prepared by a published method and purified by HPLC. On treatment with beta-glucuronidase, SPG yielded the expected amount of SA. Spectroscopic data, melting point, and optical rotation of the glucuronide and/or its triacetyl dimethyl ester derivative were consistent with the proposed structure. SPG was assayed using a 5-micron C18 column (temperature 55 degrees C) and fluorescence detection. A nonlinear gradient mobile phase at a flow rate of 2 ml/min was used, beginning with 100% 0.1 M pH 2.1 phosphate buffer and finishing with 84% buffer, 16% acetonitrile. Total run time was 25 min. Urine (10 microliter) was injected directly on the column, and quantitation was performed using urine standards. Within-run precision for SPG ranged from 1.2% at 150 mg/L to 2.4% at 5 mg/L. The limit of detection was less than 1 mg/L. A pilot study in two volunteers, each receiving a single 500-mg dose of sodium salicylate, was carried out to validate the usefulness of the assay.     

Simultaneous determination of ethylene glycol and glycolic acid in serum by gas chromatography-mass spectrometry

We describe a gas chromatographic-mass spectrometric (GC-MS) procedure for the simultaneous determination of ethylene glycol (EG) and its major toxic metabolite, glycolic acid (GA), in serum. In this method, serum (50 microL) is treated with 150 microL of glacial acetic acid/acetonitrile (1:10, v/v; contains internal standard, 1,3-propanediol, 15 mg/dL) to precipitate protein. After centrifugation, 10 microL of supernate is treated with 500 microL of 2,2-dimethoxypropane/dimethylformamide (80:20, v/v) to convert water to methanol, and the volume is then reduced to < 100 microL of dimethylformamide (but not to dryness). After formation of tertbutyldimethylsilyl derivatives, analysis is performed by capillary column GC-MS in selected ion mode. The method gives a linear response to 1000 mg/L each EG and GA (16.1 mmol/L and 13.2 mmol/L, respectively) and has a lower limit of detection and a lower limit of quantitation of 10 mg/L each EG and GA (0.16 mmol/L and 0.13 mmol/L, respectively). Total assay imprecision is CV < or = 6.4% (200 and 800 mg/L EG and GA [3.2 and 12.9 mmol/L EG; 2.6 and 10.5 mmol/L GA, respectively]). Absolute recovery from human serum was 91.1% for EG and 77.6% for GA. The procedure is free from any known interference. A complete analysis set (three calibrators, patient serum neat, patient serum diluted 1:5 (v/v), and two controls) may be completed in about 2 h. A preliminary result, based on a single calibrator and patient serum diluted 1:5 (v/v), is complete in about 1 h. The method has been used to aid the diagnosis and management in 34 cases of EG intoxication. Selected cases are briefly reviewed.     

Optimized procedure for lamotrigine analysis in serum by high-performance liquid chromatography without interferences from other frequently coadministered anticonvulsants

The authors have developed a simple isocratic high-pressure liquid chromatographic (HPLC) assay for the simultaneous determination of lamotrigine and other frequently coadministered antiepileptic drugs in serum samples. Lamotrigine extraction was performed on a reversed-phase Oasis HBL preparation column. The eluates containing butalbital as internal standard were separated with a 7-microm Chromsystems C18 250 x 4.0 mm I.D. reversed-phase column at a temperature of 40 degrees C using a mobile phase consisting of pH 3.8 phosphate-acetonitrile buffer (55:45, v/v), at a flow rate of 0.8 mL/min. Ultraviolet detection was carried out at 210 nm. Measurement of the peak:height ratio allowed quantitative determination of the samples. The method was linear over a concentration range of 0.2 to 20 microg/mL for lamotrigine. Recovery was >90%. Within-day and between-day coefficients of variation ranged from 1.8% to 6.7%. The mean lamotrigine concentration was 8.01 +/- 5.63 microg/mL. After studying sera from 130 patients treated with lamotrigine the authors confirmed that associated antiepileptic therapy affected the serum lamotrigine levels, which were significantly higher in patients under valproic acid treatment.     

High performance liquid chromatographic determination of platinum in blood and urine samples of cancer patients after administration of cisplatin drug using solvent extraction and N,N'-bis(salicylidene)-1,2-propanediamine as complexation reagent

A high performance liquid chromatographic (HPLC) procedure has been developed for the determination of cisplatin, based on the pre-column derivation of platinum(II) with reagent N,N'-bis(salicylidene)-1,2-propanediamine (H2SA2pn). The neutral platinum complex was extracted, concentrated in an organic solvent and then injected (5 microl) on a reverse phase HPLC column, Varian Micro-Pak SP C-18, 5 microm (150 mm x 4.0 mm i.d.). The complex was eluted isocratically using a ternary mixture of methanol/acetonitrile/water (40/30/30, v/v/v) at a flow rate of 1.0 ml/min and was determined by a UV detector set at 254 nm after elution. A detection limit was found to be 4.0 ng per injection. The amounts of platinum in blood serum and urine of cancer patients after administration of cisplatin were observed in a range of 221-298 ng/ml and 43-97 ng/ml with relative standard deviation (R.S.D.) of 3.6-4.6% and 3.5-4.8%, respectively. Preliminary metabolism profiles of Pt concentrations in blood and urine from the patients were established.     

Determination of pioglitazone hydrochloride in bulk and pharmaceutical formulations by HPLC and MEKC methods

High Performance Liquid Chromatographic (HPLC) and Micellar Electrokinetic Chromatographic (MEKC) methods have been developed for the determination of pioglitazone, a new englycemic antidiabetic agent. Pioglitazone and its unsaturated impurity were separated by MEKC in less than 7 min using a 43 cm x 50 microm i.d. uncoated fused-silica capillary with extended light path for better sensitivity (25 kV at 30 degrees C) and a background electrolyte (BGE) consisting of 20% acetonitrile (v/v) in 20 mM sodium borate buffer pH 9.3 containing 50 mM sodium dodecyl sulphate (SDS). The influence of various parameters on the separation such as pH of the buffer, SDS concentration, buffer concentration, organic modifiers, temperature and voltage were investigated. The MEKC method was compared with HPLC method using a 5 microm symmetry C18 column (250 x 4.6 mm i.d.) eluted with a mobile phase consisting of a mixture of 50% (v/v) acetonitrile and 10 mM potassium dihydrogen phosphate buffer, adjusting the pH to 6.0 with 0.1 M KOH. The HPLC method is capable of detecting all process related compounds, which may be present at trace levels in finished products. Both methods were fully validated and a comparison was made. The results confirm that the methods are highly suitable for its intended purpose.     

Stability of ertapenem in aqueous solutions

The kinetics of degradation of ertapenem was studied in aqueous solutions at 303, 313, 323 and 333 K and pH 0.42-12.5. Degradation was studied using two methods: HPLC (LiChrospher RP-18 column, 5 microm, 250 mm x 4 mm; mobile phase: methanol-phosphate buffer 25 mmol l(-1), pH 6.5 (15:85, v/v); flow rate--1.2 ml/min; detection UV--298 nm) and UV (294 nm). Specific acid-base catalysis involves: (a) hydrolysis of ertapenem, catalysed by hydrogen ions; (b) hydrolysis of ertapenem dianions catalysed by hydroxide ions; (c) spontaneous hydrolysis of zwitter ions and dianions of ertapenem under the influence of water. The thermodynamic parameters of these reactions--energy, enthalpy and entropy of activation were calculated. It was observed that buffer catalysis occurred in acetate, phosphate and borate buffers.     

A rapid procedure for the monitoring of amiodarone and N-desethylamiodarone by HPLC-UV detection

This article describes a rapid isocratic high-performance liquid chromatographic (HPLC) method for the simultaneous measurement of the antiarrhythmic drug amiodarone and its potentially active metabolite N-desethylamiodarone (DEA). Following a simple liquid-liquid extraction, amiodarone and its metabolite are quantitated (0.3-6.0 mg/L) by analysis on an HPLC-UV system. The analytical time was reduced by 50%, without compromising the assay performance, when Rocket column technology was employed. The assay's limit of quantitation, linearity, imprecision, and accuracy adequately covered the therapeutic range for appropriate patient monitoring. Amiodarone and DEA can be simultaneously and accurately quantitated in serum or plasma by HPLC-UV detection with imprecision < 6% at therapeutic concentrations and a quantitation range from 0.3 to 6.0 mg/L. Monitoring of this drug can allow for effective use, while minimizing serious side effects.     

Simultaneous determination of bromvalerylurea, bromodiethylacetylurea, and allylisopropylacetylurea in serum and urine by high-performance liquid chromatography with a multiwavelength UV detector and thin-layer chromatography.

A method for rapid detection and identification of bromvalerylurea (BVU), bromodiethylacetylurea (BDU), and allylisopropylacetylurea (AIU) in serum and urine by high-performance liquid chromatography (HPLC) with a multiwavelength UV detector after Sep-Pak C18 cartridge extraction is reported. A Jasco Finepak C18 reversed-phase column was used for the separation. Acetonitrile-distilled water (1:1, v/v) was used as a mobile phase. There was no significant absorption of the three hypnotics in the UV spectra (210-350 nm). However, the absorption of each was higher at the shorter wavelengths. The quantifications for the three hypnotics detected at 210 nm by the chromatogram were linear over the range 0.2-4 micrograms/mL and the detection limits of BVU, BDU, and AIU were 5, 10, and 10 ng as absolute amounts, respectively. The mean recovery yields of BVU, BDU, and AIU by Sep-Pak C18 cartridge extraction were 85.7 +/- 4.1, 98.6 +/- 2.2, and 95.1 +/- 3.5% (n = 5) in serum and 79.5 +/- 3.8, 95.7 +/- 1.8, and 93.0 +/- 4.2% (n = 5) in urine, respectively. An optimal system of thin-layer chromatography for the identification of the hypnotics is also discussed.     

Determination of gemcitabine and its metabolite in human plasma using high-pressure liquid chromatography coupled with a diode array detector

AIM:Toestablishahighpressureliquidchromatography(HPLC)methodfordeterminationoftheconcentrationofgemcitabine(dFdC)anditsmetabolite(dFdU)inhumanplasma.METHODS:Plasma1.0mLspikedwithfloxuridineasaninternalstandardwasextractedwith3.0mLofmethanol-acetonitrile(v/v,1:9).Thesupernatantwasevapo-ratedat60oCandtheresiduewasreconstitutedwith0.5mLofthesolutionusedasthemobilephase.Aftercentrifugation,50μLofthesupernatantwasinjectedintotheHPLCsystem.SeparationwasachievedonaC18(4.6mm×250mm,5μm)c…