Quantitative Colorimetric Sensing of Carbidopa in Anti-Parkinson Drugs Based on Selective Reaction with Indole-3-Carbaldehyde.

The quality of life of patients affected by Parkinson's disease is improved by medications containing levodopa and carbidopa, restoring the dopamine concentration in the brain. Accordingly, the affordable quality control of such pharmaceuticals is very important. Here is reported the simple and inexpensive colorimetric quantification of carbidopa in anti-Parkinson drugs by the selective condensation reaction between the hydrazine group from carbidopa and the formyl functional group of selected aldehydes in acidified hydroalcoholic solution. An optical assay was developed by using indole-3-carbaldehyde (I3A) giving a yellow aldazine in EtOH:H2O 1:1 (λmax~415 nm) at 70 °C for 4 h, as confirmed by LC-MS analysis. A filter-based plate reader was used for colorimetric data acquisition, providing superior results in terms of analytical performances for I3A, with a sensitivity ~50 L g-1 and LOD ~0.1 mg L-1 in comparison to a previous study based on vanillin, giving, for the same figures of merit values, about 13 L g-1 and 0.2-0.3 mg L-1, respectively. The calibration curves for the standard solution and drugs were almost superimposable, therefore excluding interference from the excipients and additives, with very good reproducibility (avRSD% 2-4%) within the linear dynamic range (10 mg L-1-50 mg L-1).

Application of net analyte signal and principal component regression for rapid simultaneous determination of Levodopa and carbidopa in commercial pharmaceutical formulation and breast (human) milk sample using spectrophotometric method.

In this study, a UV-vis spectrophotometric method coupled with net analyte signal (NAS) and principal component regression (PCR) as multivariate calibration methods were used for the simultaneous determination of levodopa (LEV) and carbidopa (CBD) in prepared mixtures, pharmaceutical formulation, and breast milk sample. The mean recovery of the NAS model was 98.10% and 99.60% for LEV and CBD, respectively. Also, the relative standard deviation (RSD%) values were found to be lower than 5.5% and 4% for LEV and CBD, respectively. On the other hand, the mean recovery of LEV and CBD related to the PCR method was obtained at 96.86% and 92.43%, respectively. K-Fold cross-validation was used to estimate the number of components, which was 7 and 3 with a mean square error prediction (MSEP) of 1.50 and 7.14 for LEV and CBD, respectively. The results revealed that the NAS model was better than the PCR model. Additionally, the proposed NAS-based calibration method was successfully developed for the simultaneous analyses of LEV and CBD in a commercial tablet and breast milk.

Melanochrome-based colorimetric assay for quantitative detection of levodopa in co-presence of carbidopa and its application to relevant anti-Parkinson drugs.

In this paper is reported the selective detection and quantification of levodopa in co-presence of carbidopa. The method took advantage of the spontaneous oxidation and color development of levodopa at basic pH here driven by alkaline earth cations and co-solvent in solution. We have shown for the first time the generation and stabilization of the purple melanochrome from levodopa, by using magnesium acetate and dimethyl sulfoxide, which was here exploited for the development of a quantitative colorimetric assay for the active principle ingredient in commercial drugs for the treatment of Parkinson's disease. The calibration curves of levodopa in the two tablet formulations, containing carbidopa as decarboxylase inhibitor, showed a common linear trend between 10 mg L-1 and 40 mg L-1 with levodopa alone or in combination with carbidopa in standard solutions, with very good reproducibility (CVav%, 3.3% for both brand and generic drug) and very good sensitivity, with limit of quantification about 0.6 mg L-1 in any case. The colorimetric method here developed is very simple and effective, appearing as a rapid and low-cost alternative to other methodologies, involving large and expensive instrumentations, for drug estimation and quality control of pharmaceutical formulations.

A rapid liquid chromatography/tandem mass spectrometry method for simultaneous determination of levodopa, carbidopa, entacapone and their six related compounds in film-coated tablets.

RATIONALE: A liquid chromatography/tandem mass spectrometry (LC/MS/MS) method has been developed and validated to determine levodopa, carbidopa, entacapone, and corresponding six related substances - levodopa impurity B, levodopa impurity C, methyldopa, methylcarbidopa, entacapone impurity C, and entacapone impurity A - in film-coated tablets for the first time. METHODS: Chromatographic separation was achieved with a gradient elution by using a C18 column, a mobile phase containing 0.5% formic acid in water and 0.5% formic acid in methanol. The mobile phase flow rate was 0.5 mL min-1 . The UV detector was set at 280 nm and the triple quadrupole mass spectrometer was used in multiple reaction monitoring (MRM) mode. RESULTS: The limit of detection (LOD) and limit of quantification (LOQ) results were 1.3 and 3.94 ng mL-1 for levodopa impurity B; 5.26 and 15.9 ng mL-1 for levodopa impurity C; 0.833 and 2.53 ng mL-1 for methyldopa; 3.31 and 10.0 ng mL-1 for methylcarbidopa; 1.67 and 5.06 ng mL-1 for entacapone impurity C; and 0.61 and 1.86 ng mL-1 for entacapone impurity A. CONCLUSIONS: The method was rapid, linear, accurate, and reproducible. The LC/MS/MS method that was developed to determine the related substances and assay of levodopa, carbidopa, and entacapone can be used to evaluate the quality of regular samples in the pharmaceutical industry. It can be also used to test the stability of samples.

3D cloves bud like Gd doped ZnO strewn rGO hybrid for highly selective determination of l-dopa in the presence of carbidopa and ascorbic acid.

An electrochemical sensor using three dimensional (3D) cloves bud like gadolinium doped ZnO nanoflowers strewn reduced graphene oxide (GZO@rGO) modified glassy carbon electrode was proposed for the sensitive and selective detection of l-dopa. The GZO@rGO nanocomposite was synthesized by hydrothermal method and characterized by a variety of analytical and spectroscopy techniques, viz. Field Emission Scanning Electron Microscopy, X-Ray Diffraction, Fourier Transformed Infrared Spectrum and X-ray photoelectron spectroscopy. The electrochemical characterization was evaluated by Cyclic Voltammetry (CV), Electrochemical Impedance Spectroscopy (EIS) and Differential Pulse Voltammetry (DPV). The 3D cloves bud like GZO@rGO hybrid displayed the highest electro-catalytic behaviour for the selective l-dopa detection. Under optimum conditions, the oxidation current response of l-dopa is directly proportional to its concentration ranging from 10 to 100 nM. The sensitivity and limit of detection was calculated as 0.1 µA nM-1 cm-2 and 0.82 nM respectively. Moreover, the proposed electrode offers excellent selectivity, because it can efficiently evade the intervention of carbidopa and ascorbic acid even in the higher concentration. Thus, the reported sensor exhibits accurate determination of l-dopa (in the presence of carbidopa & ascorbic acid) and possesses an excellent real-time application with Mucuna prurita, pharmaceutical and human urine samples.

Trypsin mediated one-pot reaction for the synthesis of red fluorescent gold nanoclusters: Sensing of multiple analytes (carbidopa, dopamine, Cu2+, Co2+ and Hg2+ ions).

Herein, we fabricated fluorescent gold nanoclusters (Au NCs) by using trypsin as a ligand. The fabricated trypsin-Au NCs emit bright red color fluorescence upon the exposure of 365 nm UV light. The trypsin-Au NCs are stable and well dispersed in water, which exhibited strong red emission peak at 665 nm upon excitation wavelength of 520 nm. The red fluorescence of trypsin-Au NCs was greatly quenched by the addition of multiple analytes such as drugs (carbidopa and dopamine) and three divalent metal ions (Cu2+, Co2+ and Hg2+ ion). As a result, a novel fluorescence "turn-off" probe was developed for the detection of the above analytes with good selectivity and sensitivity. This method exhibits the detection limits for carbidopa, dopamine, Cu2+, Co2+ and Hg2+ ions are 6.5, 0.14, 5.2, 0.0078, and 0.005 nM, respectively. The trypsin-Au NCs were successfully applied to detect drugs (carbidopa, and dopamine) in pharmaceutical samples and metal ions (Cu2+, Co2+ and Hg2+ ion) in biofluids and water samples, exhibiting good precision and accuracy, which offers a facile analytical strategy for assaying of the above analytes in pharmaceutical and biological samples.

Micellar HPLC-UV method for the simultaneous determination of levodopa, carbidopa and entacapone in pharmaceuticals and human plasma.

A method based on micellar liquid chromatography to quantify levodopa, carbidopa and entacapone in plasma is reported. The sample pretreatment was a simple dilution in a pure micellar solution then filtration and direct injection, without requiring extraction or purification steps. The three drugs were resolved from the matrix in 7 min, using an aqueous solution of 0.1 M sodium dodecyl sulphate-10% n-propanol-0.3 tiethylamine, adjusted at pH 2.8 with 0.02 M orthophosphoric acid as mobile phase, running under isocratic mode at 1.0 mL/ min through VP-ODS column. The detection was done by UV (ultraviolet) absorbance at 225 nm. The method was successfully validated by the International Conference Harmonization guidelines in terms of: selectivity, linearity (r2 > 0.998) over the concentration ranges of 0.025-1.2, 0.05-1.0 and 0.3-2.0 µg mL-1 with limits of detection of 0.01, 6.16 × 10-3 and 0.02 µg mL-1 and limits of quantification of 0.03, 0.02 and 0.07 µg mL-1 for levodopa, carbidopa and entacapone, respectively. The proposed method was applied successfully for quantification of the studied drugs in their different dosage forms. Moreover, the method was further extensive to the quantification of the studied drugs in spiked human plasma and was successfully validated by the guidelines of the European Medicines Agency. The proposed procedures were successfully evaluated to determine the studied drugs in real human plasma. The procedure was found reliable, practical, cost-effective, available, short period, easy-to-handle, low-cost, environmental-friendly, secure, useful for the analysis of numerous samples per day. Lastly, the method was performed to the analysis of incurred, using quality control samples in the same analytical run, with adequate results. Therefore, it can be implementable for custom analysis in clinical laboratories.

Improved Bioavailability of Levodopa Using Floatable Spray-Coated Microcapsules for the Management of Parkinson's Disease.

Oral administration of levodopa (LD) is the gold standard in managing Parkinson's disease (PD). Although LD is the most effective drug in treating PD, chronic administration of LD induces levodopa-induced dyskinesia. A continuous and sustained provision of LD to the brain could, therefore, reduce peak-dose dyskinesia. In commercial oral formulations, LD is co-administrated with an AADC inhibitor (carbidopa) and a COMT inhibitor (entacapone) to enhance its bioavailability. Nevertheless, patients are known to take up to five tablets a day because of poor sustained-releasing capabilities that lead to fluctuations in plasma concentrations. To achieve a prolonged release of LD with the aim of improving its bioavailability, floatable spray-coated microcapsules containing all three PD drugs were developed. This gastro-retentive delivery system showed sustained release of all PD drugs, at similar release kinetics. Pharmacokinetics study was conducted and this newly developed formulation showed a more plateaued delivery of LD that is void of the plasma concentration fluctuations observed for the control (commercial formulation). At the same time, measurements of LD and dopamine of mice administered with this formulation showed enhanced bioavailability of LD. This study highlights a floatable, sustained-releasing delivery system in achieving improved pharmacokinetics data compared to a commercial formulation.

Development and Validation of a Robust and Efficient HPLC Method for the Simultaneous Quantification of Levodopa, Carbidopa, Benserazide and Entacapone in Complex Matrices.

PURPOSE: A variety of fixed-dose combination products is used in the therapy of Parkinson Disease. However, to date a proper analytical method applicable for comparative screening of different antiparkinson products was not available. The objective of the present work was thus to develop and validate an analytical method for the simultaneous quantification of levodopa, carbidopa, benserazide and entacapone. The method should be applicable for quantifying samples from drug release experiments with marketed products and prototype formulations performed under compendial and biorelevant test conditions. METHODS: A fast and robust method applicable for separation and quantification of the four compounds was developed and validated according to International Conference on Harmonization guidelines. Method validation covered applicability to a wide concentration range of all compounds and peak separation in complex sample matrices such as biorelevant dissolution media. RESULTS: The compounds were successfully separated by using a gradient elution method on an endcapped LiChrospher 100 RP-18 (250 x 4.6 mm, 5 µm) column coupled with a LiChrospher 100 RP-18 precolumn (4 x 4 mm, 5 µm) at a column temperature of 35.0 °C and a flow rate of 1.50 mL/min. The injection volume was 30 µL and the detection wavelengths were 280 and 210 nm, respectively. For all drug/media combinations the method was linear (r2 > 0.999) for a concentration range corresponding to 1.25 - 125 % label claim (i.e. 200 mg levodopa/entacapone and 50 mg carbidopa/benserazide) released. All other validation parameters were in the specified limits over the same concentration range. CONCLUSION: The new method allows for robust and fast separation of levodopa, carbidopa, benserazide and entacapone without any interference caused by excipients or ingredients of compendial and biorelevant dissolution media and thus presents a valuable tool in both formulation development and in vitro drug release screening of numerous fixed-dose combinations of antiparkinson drugs. This article is open to POST-PUBLICATION REVIEW. Registered readers (see "For Readers") may comment by clicking on ABSTRACT on the issue's contents page.

Quantitation of levodopa and carbidopa in rat plasma by LC-MS/MS: The key role of ion-pairing reversed-phase chromatography.

A simple and selective bioanalytical method was developed for simultaneous determination of levodopa and carbidopa in rat plasma by LC-MS/MS. Levodopa and carbidopa are small polar molecules, posing challenges in the development of selective and efficient chromatography conditions. Perfluoropentanoic acid (PFPA), a volatile ion-pairing agent, was utilized to enhance chromatographic characteristics of both compounds in the reversed-phase mechanism. The ion-pairing chromatography played an essential role in mitigating matrix effects and achieving adequate separation between interfering background peaks and those of the analytes of interest, especially for levodopa. A 96-well based, automated liquid-liquid extraction, via the use Hamilton NIMBUS liquid handlers, was developed. Butyl alcohol, when mixed with ethyl acetate, greatly increased the recovery of both levodopa and carbidopa. The addition of PFPA further enhanced recovery for both analytes. Sodium metabisulfite, an antioxidant, was used to stabilize levodopa and carbidopa in rat plasma. The method was validated in the ranges of 50-10,000ng/mL and 25-5000ng/mL for levodopa and carbidopa, respectively, using levodopa-d3 and carbidopa-d3 as internal standards. The validated method was successfully applied to analyze rat plasma samples from in-life studies.

Investigation of different spectrophotometric and chemometric methods for determination of entacapone, levodopa and carbidopa in ternary mixture.

New, simple, accurate and sensitive UV spectrophotometric and chemometric methods have been developed and validated for determination of Entacapone (ENT), Levodopa (LD) and Carbidopa (CD) in ternary mixture. Method A is a derivative ratio spectra zero-crossing spectrophotometric method which allows the determination of ENT in the presence of both LD and CD by measuring the peak amplitude at 249.9nm in the range of 1-20µgmL-1. Method B is a double divisor-first derivative of ratio spectra method, used for determination of ENT, LD and CD at 245, 239 and 293nm, respectively. Method C is a mean centering of ratio spectra which allows their determination at 241, 241.6 and 257.1nm, respectively. Methods B and C could successfully determine the studied drugs in concentration ranges of 1-20µgmL-1 for ENT and 10-90µgmL-1 for both LD and CD. Methods D and E are principal component regression and partial least-squares, respectively, used for the simultaneous determination of the studied drugs by using seventeen mixtures as calibration set and eight mixtures as validation set. The developed methods have the advantage of simultaneous determination of the cited components without any pre-treatment. All the results were statistically compared with the reported methods, where no significant difference was observed. The developed methods were satisfactorily applied to the analysis of the investigated drugs in their pure form and in pharmaceutical dosage forms.

Sensitive determination of carbidopa through the electrochemiluminescence of luminol at graphene-modified electrodes.

Using the concept of electrogenerated chemiluminescence (ECL), a sensitive analytical method for the determination of carbidopa is described. Electro-oxidation of carbidopa on the surface of a graphene oxide (GO)-modified gold electrode (GE) leads to enhancement of the weak emission of oxidized luminol. Under optimum experimental conditions, the ECL signal increases linearly with increasing carbidopa concentrations over a range of 1.0 × 10(-9) -1.7 × 10(-7) M, with a detection limit of 7.4 × 10(-10) M. The proposed ECL method was successfully used for the determination of carbidopa in urine samples.

Synthesis of ZnO nanorods and their application in the construction of a nanostructure-based electrochemical sensor for determination of levodopa in the presence of carbidopa.

A novel carbon paste electrode modified with ZnO nanorods and 5-(4'-amino-3'-hydroxy-biphenyl-4-yl)-acrylic acid (3,4'-AAZCPE) was fabricated. The electrochemical study of the modified electrode, as well as its efficiency for the electrocatalytic oxidation of levodopa, is described. The electrode was employed to study the electrocatalytic oxidation of levodopa, using cyclic voltammetry (CV), chronoamperometry (CHA), and square-wave voltammetry (SWV) as diagnostic techniques. It has been found that the oxidation of levodopa at the surface of the modified electrode occurs at a potential of about 370 mV less positive than that of an unmodified carbon paste electrode. The SWV results exhibit a linear dynamic range from 1.0 × 10(-7) M to 7.0 × 10(-5) M and a detection limit of 3.5 × 10(-8) M for levodopa. In addition, this modified electrode was used for the simultaneous determination of levodopa and carbidopa. Finally, the modified electrode was used for the determination of levodopa and carbidopa in some real samples.

Simultaneous estimation of levodopa and carbidopa by RP-HPLC using a fluorescence detector: its application to a pharmaceutical dosage form.

A reverse-phase high-performance liquid chromatographic (RP-HPLC) method was developed and validated for the simultaneous estimation of levodopa and carbidopa in bulk and pharmaceutical formulations. Chromatographic separation was achieved by using a C18 reverse-phase column and a mixture of an aqueous phase (10 mM potassium dihydrogen phosphate buffer, pH4.0) and methanol (90:10 v/v) as the mobile phase. Quantitative analysis of levodopa and carbidopa was performed using a fluorescence detector at an excitation wavelength of 280 nm and an emission wavelength of 310 nm. The method was linear between 5 and 500 ng/mL for both levodopa and carbidopa. The detection limits for levodopa and carbidopa were 0.30 and 0.60 ng/mL, respectively, whereas the quantitation limit was 0.80 ng/mL for levodopa and 1.2 ng/mL for carbidopa. The method demonstrated good and consistent recoveries (99.63-100.80% for levodopa and 98.97-100.94% for carbidopa) with low interday and intraday relative standard deviation. The validated method was successfully applied to quantify levodopa and carbidopa simultaneously in a pharmaceutical formulation. The method was found to be precise, sensitive and accurate for the simultaneous determination levodopa and carbidopa in bulk and pharmaceutical formulations.

Voltammetric detection of L-dopa and carbidopa on graphene modified glassy carbon interfaces.

The present work reports on the use of graphene nanosheets, deposited on glassy carbon, as electrode materials, and their electrochemical characterization. The graphene nanosheets were obtained by chemical reduction of graphene oxide using hydrazine. The possibility of analyzing L-dopa and carbidopa, two important catecholamines found in pharmaceutical products, separately and simultaneously by differential pulse voltammetry utilizing graphene modified GC interfaces is investigated. Voltammetric peak currents showed a linear response for both catecholamines in the range of 1-16 µM. The detection limit was about two times lower for L-dopa than carbidopa being 0.8 µM and 1.8 µM, respectively with a current sensitivity of (2.15 ± 0.5) and (0.48 ± 0.3) µA µM(-1). Simultaneous detection of both catecholamines can be achieved on these electrodes. Equivalent amounts of L-dopa and carbidopa have no effect on the detection limit of L-dopa. In addition, the presence of L-dopa with concentrations 4 times higher than carbidopa has no influence on the voltammetric profile.

Application of nanosized MCM-41 to fabrication of a nanostructured electrochemical sensor for the simultaneous determination of levodopa and carbidopa.

A novel carbon paste electrode modified with nanosized mesoporous MCM-41 was prepared, and used as an electrochemical sensor to study the electro oxidation of levodopa (LD), carbidopa (CD) and their mixtures. Using differential pulse voltammetry (DPV), a highly selective and simultaneous determination of LD and CD has been explored at the modified electrode. The electrochemical sensor displayed a good resolving function for the overlapping voltammetric responses of LD and CD into two separate peaks with a potential difference of 370 mV. DPV peak currents of LD increased linearly with concentration over the 0.13 µM to 1250.00 µM range and exhibited a detection limit of 0.072 µM. Also, the proposed electrochemical sensor was used for the determination of LD and CD in some real samples, using the standard addition method.

Electrocatalytic oxidation and voltammetric determination of levodopa in the presence of carbidopa at the surface of a nanostructure based electrochemical sensor.

In the present paper, the use of a carbon paste electrode modified by meso-tetrakis(3-methylphenyl) cobalt porphyrin (CP) and TiO(2) nanoparticles for the determination of levodopa (LD) and carbidopa (CD) was described. Initially, cyclic voltammetry was used to investigate the redox properties of this modified electrode at various scan rates. Next, the mediated oxidation of LD at the modified electrode was described. At the optimum pH of 7.0, the oxidation of LD occurs at a potential about 150 mV less positive than that of an unmodified carbon paste electrode. Based on differential pulse voltammetry (DPV), the oxidation of LD exhibited a dynamic range between 0.1 and 100.0 µM and a detection limit (3σ) of 69 ± 2 nM. DPV was used for simultaneous determination of LD and CD at the modified electrode, and quantitation of LD and CD in some real samples (such as tablets of Parkin-C Fort and Madopar, water, urine, and human blood serum) by the standard addition method.

Simultaneous voltammetric determination of levodopa, carbidopa and benserazide in pharmaceuticals using multivariate calibration.

An analytical methodology based on differential pulse voltammetry (DPV) on a glassy carbon electrode and the partial least-squares (PLS-1) algorithm for the simultaneous determination of levodopa, carbidopa and benserazide in pharmaceutical formulations was developed and validated. Some sources of bi-linearity deviation for electrochemical data are discussed and analyzed. The multivariate model was developed as a ternary calibration model and it was built and validated with an independent set of drug mixtures in presence of excipients, according with manufacturer specifications. The proposed method was applied to both the assay and the uniformity content of two commercial formulations containing mixtures of levodopa-carbidopa (10:1) and levodopa-benserazide (4:1). The results were satisfactory and statistically comparable to those obtained by applying the reference Pharmacopoeia method based on high performance liquid chromatography. In conclusion, the methodology proposed based on DPV data processed with the PLS-1 algorithm was able to quantify simultaneously levodopa, carbidopa and benserazide in its pharmaceuticals formulations using a ternary calibration model for these drugs in presence of excipients. Furthermore, the model appears to be successful even in the presence of slight potential shifts in the processed data, which have been taken into account by the flexible chemometric PLS-1 approach.

Kinetic approach for the enzymatic determination of levodopa and carbidopa assisted by multivariate curve resolution-alternating least squares.

A combination of kinetic spectroscopic monitoring and multivariate curve resolution-alternating least squares (MCR-ALS) was proposed for the enzymatic determination of levodopa (LVD) and carbidopa (CBD) in pharmaceuticals. The enzymatic reaction process was carried out in a reverse stopped-flow injection system and monitored by UV-vis spectroscopy. The spectra (292-600 nm) were recorded throughout the reaction and were analyzed by multivariate curve resolution-alternating least squares. A small calibration matrix containing nine mixtures was used in the model construction. Additionally, to evaluate the prediction ability of the model, a set with six validation mixtures was used. The lack of fit obtained was 4.3%, the explained variance 99.8% and the overall prediction error 5.5%. Tablets of commercial samples were analyzed and the results were validated by pharmacopeia method (high performance liquid chromatography). No significant differences were found (alpha=0.05) between the reference values and the ones obtained with the proposed method. It is important to note that a unique chemometric model made it possible to determine both analytes simultaneously.

Quantitative analysis of levodopa, carbidopa and methyldopa in human plasma samples using HPLC-DAD combined with second-order calibration based on alternating trilinear decomposition algorithm.

An HPLC method combined with second-order calibration based on alternating trilinear decomposition (ATLD) algorithm has been developed for the quantitative analysis of levodopa (LVD), carbidopa (CBD) and methyldopa (MTD) in human plasma samples. Prior to the analysis of the analytes by ATLD algorithm, three time regions of chromatograms were selected purposely for each analyte to avoid serious collinearity. Although the spectra of these analytes were similar and interferents coeluted with the analytes studied in biological samples, good recoveries of the analytes could be obtained with HPLC-DAD coupled with second-order calibration based on ATLD algorithm, additional benefits are decreasing times of analysis and less solvent consumption. The average recoveries achieved from ATLD with the factor number of 3 (N=3) were 100.1+/-2.1, 96.8+/-1.7 and 104.2+/-2.6% for LVD, CBD and MTD, respectively. In addition, elliptical joint confidence region (EJCR) tests as well as figures of merit (FOM) were employed to evaluate the accuracy of the method.