Comparative analysis of the full set of methylated ß-cyclodextrins as chiral selectors in capillary electrophoresis.

The chiral separation ability of the full library of methylated-ß-cyclodextrins towards pharmacologically significant racemic drugs including basic compounds was studied by chiral CE. The syntheses of all the methylated, single isomer ß-cyclodextrins were revised and optimized and the aqueous solubility of the derivatives was unambiguously established. The three most relevant commercially available methylated isomeric mixtures were also included in the screening, so a total of ten various methylated CDs were investigated. The effects of the selector concentration on the enantiorecognition properties at acidic pH were investigated. Among the dimethylated ß-cyclodextrins, the heptakis (2,6-di-O-methyl)-ß-cyclodextrin isomer (2,6-DIMEB) resulted to be the most versatile chiral selector. Terbutaline was selected as a model compound for the in-depth investigation of host-guest enantiodiscrimination ability. The association constants between the two terbutaline enantiomers and 2,6-DIMEB were determined in order to support that the enantioseparation is driven by differences is host-guest binding. The migration order of the enantiomers was confirmed by performing spiking experiments with the pure enantiomers. 1D and 2D NMR spectroscopy was applied to the 2,3-, and 2,6-DIMEB/terbutaline systems to rationalize at molecular level the different enantioseparation ability of the dimethylated ß-cyclodextrin selectors.

Spectrofluorimetric investigation with green analytical procedures for estimation of bambuterol and terbutaline: Application to pharmaceutical dosage forms and content uniformity testing.

Bambuterol (BAM) and terbultaline (TER) are well known and effective bronchodilators. In this article highly sensitive, green and cost-effective spectrofluorimetric methods are designed to determine low concentrations of such drugs. The proposed methods are based on an investigation of the native fluorescence properties of aqueous solutions of BAM at 298 nm after excitation at 263 nm and of TER at 313 nm after excitation at 275 nm. Under optimum conditions, the plots of the relative fluorescence intensity versus concentration were rectilinear over the range 0.1-1.2 µg/mL for BAM and 0.05-0.5 µg/mL for TER with a limit of quantitation of 0.067 µg/mL for BAM and 0.018 µg/mL for TER. The methods are simple and hence suitable for application to the quantification of BAM and TER in syrups and tablets without interference from common excipients. Furthermore, based on United States Pharmacopeia (USP) guidelines, the application was extended to determine the content uniformity of the cited drugs in low dose tablets. The developed methods were fully validated according to the guidelines of the International Conference on Harmonization (ICH).

RP-HPLC-UV method development and validation for simultaneous determination of terbutaline sulphate, ambroxol HCl and guaifenesin in pure and dosage forms.

OBJECTIVE: The objective of the present work was to develop and validate a simple, sensitive, rapid and stable reverse-phase high performance liquid chromatography (RP-HPLC) method for a combination of Terbutaline sulphate (TSL), Ambroxol hydrochloride (AML) and Guaifenesin (GFN). METHOD: The combination of these drugs was analyzed by using Shimadzu LC 2010 CHT high performance liquid chromatography (HPLC). Successful separation was achieved by isocratic elution on a reverse-phase C18 column (sun fire) (250mm, 4.6mm, 5µ), using a mobile phase consisting of buffer: acetonitrile in the ratio 80: 20 (buffer - 0.1% v/v triethyleamine pH-3.0) followed by 1.0mL/min flow rate. The wavelength of detection was at 220nm. RESULT: The chromatographic retention times were consistent at 3.0, 10.5 and 13.8minutes for TSL, AML and GFN respectively. For these three compounds, the lower limit of detection was 1.0, 1.25, and 1.5µg/mL and lower limit of quantification was 3.3, 4.1 and 5.0µg/mL respectively. The linearity concentrations established for TSL, AML and GFN were 1.0-7.0, 1.5-7.5 and 4.0-14.0µg/mL respectively. The correlation coefficients for all the drugs were found to be greater than 0.999. The relative standard deviation of inter- and intra-day were less than 2.0%. CONCLUSION: This method provides a necessary tool for quantification of the selected drugs for their assay. The proposed method is simple, accurate, reproducible and applied successfully to analyze three compounds in pure as well dosage form.

Determination of beta-agonists in swine hair by µFIA and chemiluminescence.

ß-Agonists are a group of illegal feed additives. In this paper, it was found that the light emission produced by the oxidation of luminol by potassium ferricyanide was enhanced by the ß-agonists (ractopamine, salbutamol, and terbutaline). Based on chemiluminescence phenomenon, a novel, rapid, and sensitive microflow injection analysis system on a microfluidic glass chip was established for determination of the ß-agonists. The chip was fabricated from two glass plates (64 mm × 32 mm) with microchannels of 200 µm width and 100 µm depth. The detection limits were achieved at 2.0 × 10(-8) mol/L of ractopamine, 1.0 × 10(-8) mol/L of terbutaline and 5.0 × 10(-7) mol/L of salbutamol. In this report, our method was applied for determination of the ß-agonists in swine hair from three different sources with satisfactory results.

Application of a new spectrophotometric method manipulating ratio spectra for determination of bambuterol hydrochloride in the presence of its degradation product terbutaline.

A simple, specific, accurate and precise spectrophotometric stability indicating method is developed for determination of bambuterol hydrochloride (BH) in the presence of its degradation product terbutaline (TERB) and in pharmaceutical formulations. A newly developed spectrophotometric method called ratio difference method by measuring the difference in amplitudes between 245 and 260 on of ratio spectra. The calibration curves are linear over the concentration range of 0. 1 - 1 mg . mL-1 for BH and 0. 1-0. 7 mg . mL-1 for TERB with mean percentage recovery of 100. 56 ± 0. 751 and 99. 88 ± 1. 183, respectively. The selectivity of the proposed method is checked using laboratory prepared mixtures. The proposed method has been successfully applied to the analysis of BH in pharmaceutical dosage forms without interference from other dosage form additives and the results have been statistically compared with pharmacopeial method.

Electrically assisted liquid-phase microextraction for determination of ß2-receptor agonist drugs in wastewater.

Electromembrane extraction followed by high-performance liquid chromatography coupled with ultraviolet detection was validated for the determination and quantification of salbutamol (SB) and terbutaline in aqueous samples. A 200-V electrical field was applied to extract the analytes from 2.5 mL sample solution with pH 3.0, through an organic phase which consisted of 80% 2-nitrophenyl octyl ether, 10% di-(2-ethylhexyl) phosphate and 10% tris-(2-ethylhexyl)phosphate as supported liquid membrane into an acidic acceptor solution with pH 1.0, located inside the lumen of a hollow fiber. To achieve the best extraction conditions, the organic membrane composition was optimized separately and other parameters, such as extraction time, applied voltage and pH in sample solution and acceptor phase were studied using experimental design. Under optimal conditions, extraction recoveries of 53 and 43% were obtained for SB and terbutaline, respectively, which corresponded to preconcentration factors of 89 for SB and 72 for terbutaline. The method offers acceptable linearity with correlation coefficient higher than 0.9947 and relative standard deviation less than 4.7%. Finally, it was applied for analysis of drugs in wastewater samples.

[Detection and Analysis of Drug Crystals in Medical Transdermal Patches by Using X-ray Diffraction Measurement].

The crystallization of active pharmaceutical ingredients (APIs) in matrix-type transdermal patches has implications for the rate of drug absorption through the skin and patch adhesion strength. Therefore, the presence or absence and the degree of API crystallinity must be controlled to guarantee the quality of patches. In this study, the utility of laboratory-level X-ray diffractometers for the detection and analysis of crystalline APIs in transdermal patches was investigated using medical patches of tulobuterol and isosorbide dinitrate. Several matrix-type patches employ a controlled drug delivery system containing intentionally crystallized API. Both benchtop and high-resolution laboratory X-ray diffractometers can detect several characteristic peaks of the APIs in these patches even if the patches are wrapped in an outer bag, although a benchtop model provides peak heights one-seventh to one-fifth that of a high-resolution instrument. An isosorbide dinitrate patch containing an unintentionally crystallized spot was wrapped in an outer bag, followed by measurements using both X-ray diffractometers. For both instruments, several isosorbide dinitrate-derived peaks were detected only at the crystallized spot, although the signal-to-noise ratio was poorer for the benchtop model. These results show that a high-resolution X-ray diffractometer is advantageous for high-detection sensitivity and offers a high degree of freedom of the measurement position on the sample. It was concluded that a laboratory-level high-resolution X-ray diffractometer can be used to examine the crystalline state of APIs in patches inside an unopened outer bag.

Rapid analysis of aerosol drugs using nano extractive electrospray ionization tandem mass spectrometry.

Aerosol drugs dominate a significant share of pharmaceutical preparations on the market. A novel sensitive method utilizing nano extractive electrospray ionization mass spectrometry (nanoEESI-MS) has been developed for the rapid analysis of aerosol drug samples with quantitative information. Without any sample pretreatment, aerosol drugs were manually sprayed into the primary ion plume created by a nano electrospray emitter for direct ionization under ambient conditions. The analyte ions of interest were guided into an ion trap mass spectrometer for tandem mass analysis. The active ingredients of various aerosol drugs, such as econazole nitrate, beclomethasone dipropionate, binary mixture of methyl salicylate and diphenhydramine, terbutaline, and salbutamol, were rapidly detected using nanoEESI-MS. A single sample analysis could be completed within 1.2 s. Tandem mass spectrometry was used to confirm the identification of important compounds in each aerosol drug sample. Reasonable relative standard deviation (RSD = 6.39%, n = 13) and acceptable sensitivity (10 ppt, 100 muL) were found for the salbutamol aerosol sample, which suggests that nanoEESI-MS has the quantitative capacity for analyzing complex pharmaceutical samples. This method was further extended to study the thermal decomposition process of salbutamol, showing that the degradation kinetics of salbutamol can be conveniently tracked. Our data demonstrate that nanoEESI tandem mass spectrometry is a fast and sensitive technique for the analysis of aerosol drug preparations, showing promising applications in pharmacology studies and in situ analysis of aerosol drugs on the market.

A sensitive non-aqueous capillary electrophoresis-mass spectrometric method for multiresidue analyses of beta-agonists in pork.

Non-aqueous capillary electrophoresis-mass spectrometry (NACE-MS) was developed for trace analyses of beta-agonists (i.e. clenbuterol, salbutamol and terbutaline) in pork. The NACE was in 18 mM ammonium acetate in methanol-acetonitrile-glacial acetic acid (66 : 33 : 1, v/v/v) using a voltage of 28 kV. The hyphenation of CE with a time-of-flight MS was performed by electrospray ionization interface employing 5 mM ammonium acetate in methanol-water (80 : 20, v/v) as the sheath liquid at a flow rate of 2 microL/min. Method sensitivity was enhanced by a co-injection technique (combination of hydrodynamic and electrokinetic injection) using a pressure of 50 mbar and a voltage of 10 kV for 12 s. The method was validated in comparison with HPLC-MS-MS. The NACE-MS procedure provided excellent detection limits of 0.3 ppb for all analytes. Method linearity was good (r(2) > 0.999, in a range of 0.8-1000 ppb for all analytes). Precision showed %RSDs of <17.7%. Sample pre-treatment was carried out by solid-phase extraction using mixed mode reversed phase/cation exchange cartridges yielding recoveries between 69 and 80%. The NACE-MS could be successfully used for the analysis of beta-agonists in pork samples and results showed no statistical differences from the values reported by the Ministry of Public Health, Thailand using HPLC-MS-MS method.

Determination of terbutaline sulfate by capillary electrophoresis with chemiluminescence detection.

A novel capillary electrophoresis (CE) with chemiluminescence (CL) detection method for determination of terbutaline sulfate has been developed. This method is based on the chemiluminescence reaction of potassium ferricyanide with luminol in sodium hydroxide medium sensitized by terbutaline sulfate. With the peak height as a quantitative parameter applying optimum working conditions, terbutaline sulfate is determined over the range of 7.0 x 10(-8) to 3.6 x 10(-6)M with a detection limit of 3.0 x 10(-8)M. The relative standard deviation (RSD) was 4.6% for 6.0 x 10(-7)M terbutaline sulfate (n=11). The proposed method has been applied to determination of terbutaline sulfate in commercial terbutaline sulfate drug and spiked in human urine with satisfactory results.

Chemical mapping of tulobuterol in transdermal tapes using microscopic laser Raman spectroscopy.

Microscopic Laser Raman Spectroscopy and Mapping (MLRSM) technique was used to investigate the distribution of tulobuterol (TBR) crystals in transdermal tapes. TBR is one of suitable compounds for the transdermal pharmaceuticals because it has high permeability into skin. In case of TBR transdermal tapes, some commercial products also contain TBR crystals in order to control a release rate from a matrix. Therefore, the presence of TBR crystals in the matrix is a critical factor for quality assurance of this type of TDDS tapes. The model tapes prepared here employed two kinds of matrices, i.e., rubber or acrylic, which are generally used for transdermal pharmaceuticals. TBR crystals in the matrix were observed by MLRSM. Accurate observation of the distribution of TBR in the tapes was achieved by creating a Raman chemical map based on detecting unique TBR peak in each pixel. Moreover, differences in the growth of TBR crystals in the two kinds of matrices were detected by microscopic observation. MLRSM also enabled the detection of TBR crystals in commercial products. The present findings suggest that Raman micro-spectroscopic analysis would be very useful for verifying and/or assessing the quality of transdermal pharmaceuticals in development, as well as for manufacturing process control.

Potentiometric sensor based on novel flowered-like Mg-Al layered double hydroxides/multiwalled carbon nanotubes nanocomposite for bambuterol hydrochloride determination.

Nowadays, development of highly efficient potentiometric sensors attracts the attention of many researchers over the world; due to the great expansion of portable analytical devices. This study aims to apply a current development to the construction and sense of carbon paste sensors based on flowered-like Mg-Al layered double hydroxides/multiwalled carbon nanotubes (FLLDH/MWCNTs) (sensor І), FLLDH/titanate nanotubes (TNTs) (sensor ІІ) and MWCNTs/TNTs (sensor ІІІ) nanocomposites for bambuterol hydrochloride analysis; to enhance the potentiometric response towards determination of the drug. The sensors exhibited excellent Nernstian slopes 58.8 ±â€¯0.5, 58.5 ±â€¯0.8 and 57.4 ±â€¯0.7 mV/decade with linear working ranges of 1.0 × 10-7-1.0 × 10-2, 1.0 × 10-6-1.0 × 10-2 and 1.0 × 10-7-1.0 × 10-2 mol L-1, detection limits 2.3 × 10-8, 2.5 × 10-7and 7.5 × 10-8 mol L-1 and quantification limits of 7.6 × 10-8, 8.3 × 10-7and 2.5 × 10-7 mol L-1 for sensor І, ІІ and ІІІ, respectively. The selectivity behaviour of the investigated sensors was tested against biologically important blood electrolytes (Na+, K+, Mg2+, Ca2+). The proposed analytical method was successfully applied for BAM determination in pure drug, pharmaceutical products, surface water, human plasma and urine samples with excellent recovery data (99.62, 99.10 and 98.95%) for the three sensors, respectively.

Effects of Terbutaline on Growth Performance, Carcass Quality, Some Biochemical Parameters and its Residues in Broiler Chicken.

BACKGROUND AND OBJECTIVE: Terbutaline is a ß-agonist that used as growth promoters to improved carcass chemical composition of chicks without residues. The purpose of the present investigation is exploring the effect of different dietary levels and duration of terbutaline on the productive performance, biochemical and carcass quality traits including residue of acres broiler. MATERIALS AND METHODS: A total of 150 one-day-old arbor acres broiler chicks were allotted into 5 groups (3 replicates per each). Group 1 was fed on the basal diet without supplement, while groups 2-5 fed on the basal diet supplemented by 5 or 10 mg terbutaline kg-1 diet during 1-42 or 21-42 days, respectively. RESULTS: When handling the dietary levels and duration of terbutaline, results of the present study showed that10 mg terbutaline kg-1 diet during the whole experimental period is a more effective dose for improvement of growth performance with significant (p<0.05) increased serum protein and breast muscles relative weight compared with control. Also, 10 mg terbutaline kg-1 diet during the whole experimental period significantly (p<0.05) increase d CP% (crude protein%) and CHO% (carbohydrate%) of breast muscle and significantly (p<0.05) decreased fat% (ether extract%) of breast muscle and abdominal fat relative weight compared with control. Meanwhile, 5 mg terbutaline kg-1 diet during 1-42 or 21-42 days has no significant effect on the above-mentioned parameters. Regarding residue, the terbutaline residue wasn't detected in broiler meat. CONCLUSION: It can conclude that 10 mg terbutaline kg-1 diet during the whole experimental period is a better dose and duration for improving growth performance, the chemical composition of breast muscle and carcass traits of broiler chickens with no terbutaline residue in breast muscle.

Rapid and mild fabrication of protein membrane coated capillary based on supramolecular assemble for chiral separation in capillary electrochromatography.

Exploration of the simple and stable coating methods is of great significance in capillary electrochromatography (CEC). In this work, a lysozyme assemble supramolecular membrane coated capillary column was developed for CEC chiral separation. Taking advantage of phase transformation of lysozyme, the coating process was achieved within 1.0 h thus to a large extent reduced the capillary preparation time and simplified the coating procedure. The successful fabrication of the supramolecular membrane coated capillary was verified by scanning electron microscopy (SEM), Fourier transform-infrared spectroscopy (FT-IR), fluorescence imaging, and electroosmotic flow (EOF). The separation capacity of the coated capillary was evaluated by analysis of different chiral analytes, including chiral amine drug and neurotransmitters, and good enantioseparation efficiency was achieved for the three pairs of enantiomers. For three consecutive runs, the relative standard deviations (RSD) for the migration time of the analytes for intra-day (n = 3), inter-day (n = 3) and column-to-column (n = 3) were in the range of 0.7-1.5%, 2.7-3.6%, and 4.5-5.8%, respectively. Additionally, the supramolecular membrane coated capillary column could run consecutively 100 times without observable change in the separation efficiency, proving the feasibility of the coating method based on the adhesion of the protein-based supramolecular membrane.

Multiresidue analysis of beta-agonists in bovine and porcine urine, feed and hair using liquid chromatography electrospray ionisation tandem mass spectrometry.

The use of beta-agonists as growth promoters in cattle breeding is forbidden in many countries for reasons of fair trade and consumer protection. In recent years the use of liquid chromatography (LC) tandem mass spectrometry (MS/MS) has been shown to be the method of choice for the control of beta-agonists. In this study an LC-MS/MS multiresidue analysis method is presented for trace analysis of 22 beta-agonists. A truly generic concept has been designed based on mixed-mode solid-phase extraction and positive electrospray ionisation LC-MS/MS operated in the multiple reaction monitoring mode. This method allows application to a wide variety of sample matrices such as urine, feed and hair, following minor modifications to the analysis procedure only. The method features fit-for-purpose sensitivity in urine as shown by CCalpha and CCbeta values of less than 0.2 and less than 0.5 microg/l respectively, for all beta-agonists studied (terbutaline and reproterol, less than 0.3 and less than 1.0 respectively). Similar but semiquantitative application to feed and hair showed CCbeta values of less than 10.0 and less than 5.0 microg/kg, respectively. A further simplification and improvement is demonstrated using Ultra Performance LC (UPLC) and fast-switching MS/MS. The successful validation of this method following the latest EU requirements and its application to real samples demonstrate that a new versatile tool has been achieved for veterinary control of beta-agonists.

Assuring quality of drugs by monitoring impurities.

To assure the quality of drugs, impurities must be monitored carefully. It is important to understand what constitutes an impurity and to identify potential sources of such impurities. Selective analytical methods need to be developed to monitor them. It is generally desirable to profile impurities to provide a yardstick for comparative purposes. New impurities may be observed as changes are made in the synthesis, formulation, or production procedures, albeit for improving them. At times it is necessary to isolate and characterize an impurity when hyphenated methods do not yield the structure or when confirmation is necessary with an authentic material. Availability of an authentic material can also allow toxicological studies and provide a standard for routine monitoring of the drug product.

New ipratropium formulation to decrease nebulization time.

A new anticholinergic aerosol containing 0.5mg ipratropium bromide dissolved in 1mL of solution has been produced with the purpose of decreasing nebulization time for patients compared to the traditional formulation which is twice as voluminal (0.5mg/2mL, Boehringer-Ingelheim, France). The aim of this study was to compare aerosol characteristics (inhaled mass, particle size distribution and nebulization time) of these two formulations of ipratropium bromide, nebulized alone and with terbutaline (5mg/2mL, Astra Zeneca, Sweden), to determine whether the new formulation was equivalent to the old one. Four different jet nebulizers were used: PariLC+, Atomisor NL9M, Sidestream and Mistyneb. Statistical analysis of the results showed that for all types of nebulizer, the inhaled mass of ipratropium bromide 0.5mg/1mL was significantly lower than the inhaled mass of ipratropium bromide 0.5mg/2mL, and that there was no statistical difference between the inhaled mass of ipratropium bromide 0.5mg/1mL+terbutaline 5mg/2mL and the inhaled mass of ipratropium bromide 0.5mg/2mL+terbutaline 5mg/2mL. The study also showed that the new formulation of ipratropium bromide (0.5mg/1mL) mixed with terbutaline allowed a 26% decrease in nebulization time compared to the old formulation (0.5mg/2mL) mixed with terbutaline without changing aerosol characteristics (inhaled mass and particle size distribution). This leads to the conclusion that a 2mL minimum volume is required for nebulization, and that nebulization of ipratropium bromide 0.5mg/1mL alone must be avoided.

Factorial design applied to a non-aqueous capillary electrophoresis method for the separation of beta-agonists.

The aim of this work was to study the effects of both chemical and instrumental parameters on the separation of beta-agonists (clenbuterol (CLE), salbutamol (SAL) and terbutaline (TER)) by non-aqueous capillary electrophoresis (NACE) method. Due to the number of parameters involved and their interactions, factorial experimental designs (EDs) at two levels was applied to investigate the influence of experimental factors (ionic strength of the background electrolyte (BGE), organic solvent, injection time, voltage and temperature) in sets of several CE responses (resolution, (RS), number of theoretical plate (N), tailing factor (TF) and migration time (tm)). As a compromise between the four responses, the optimum condition was obtained in 18 mM ammonium acetate in methanol (MeOH):acetonitrile (ACN):glacial acetic acid (66:33:1%, v/v/v) using an injection time of 4 s, the voltage and the temperature of 28 kV and 24 degrees C, respectively. The proposed NACE permitted the baseline separation of the three beta-agonists within 10.5 min with good repeatability (%RSD < 3.5%) and linearity (r2 > 0.99). The developed method was applicable for the analysis of the beta-agonists in syrup and tablets and the NACE condition was compatible with a mass spectrometer detector.

[Simultaneous determination of the residues of four ß2-agonists in animal foods by modified high performance liquid chromatography-tandem mass spectrometry].

An analytical method was developed to simultaneously determine the residues of four ß2-agonists in animal foods by high performance liquid chromatography/electrospray-tandem mass spectrometry (HPLC-MS/MS). Samples were extracted with 5% (mass fraction) trichloracetic acid, and then cleaned up by two solid phase extraction cartridges (HLB and ProElut PXC). Quantification of the four ß2-agonists was achieved by HPLC-MS/MS in multiple reaction monitoring (MRM) using internal standard method. The limits of detection (S/N=3) of clenbuterol, salbutamol, ractopamine and terbutaline were 0. 05, 0. 05, 0. 05 and 0. 2 µg/kg, and the limits of quantification (S/N= 10) were 0. 25, 0. 25, 0. 1 and 0. 5 µg/kg, respectively. The average recoveries of the four ß2-agonists spiked in blank samples at the spiked levels of 2. 5, 5 and 10 µg/kg were 90. 3%-120. 5% with the relative standard deviation (RSD) range of 1. 60%-9. 33%. The method is reliable, sensitive, good recovery and repeatability. It is suitable for the determination of the residues of the four ß2-agonists in animal foods.

Impact of Ternary Solvent System in Stability-Indicating Assay Method of Bambuterol: Design of Experiments Approach.

High-performance liquid chromatography method for anti-asthmatic ß2-agonist drug bambuterol, its process-related impurities and its major degradation products was developed and validated using quality by design concept. A 3(3) full factorial design was employed to study the effect of three independent factors, namely, ratio of organic modifiers in mobile phase, pH of the buffer and flow rate of the mobile phase. The responses considered were retention time of the last peak and resolution of poorly separated peaks (drug and PR-4 and drug and DP-3). The optimum conditions for separation were determined with the aid of design of experiments. The optimized ternary solvent composition was a mixture of 10 mM ammonium acetate buffer (pH 6.0), methanol and acetonitrile in the ratio of 90:5: 5 (v/v/v) in solvent reservoir A and 10:45:45 (v/v/v) in solvent reservoir B. The separation of the analytes was achieved by using a gradient method. The predictability criteria of the optimized method demonstrated good correlation between observed and predicted response. The method was validated for specificity, linearity, accuracy, precision and robustness in compliance with the International Conference on Harmonization guidelines Q2R1.