INTRODUCTION:

Stroke is characterized by the sudden loss of blood circulation to an area of the brain, resulting in a corresponding loss of neurologic function. Strokes are classified as either hemorrhagic or ischemic. Acute ischemic stroke caused by thrombosis or embolism. The term ischemic stroke is used to describe a variety of conditions in which blood flow to part or all of the brain is reduced, resulting in tissue damage.

Edaravone and Argatroban combination was approved on 13 September 2002, Patented by Mitsubishi Tanabe Pharma Corporation, Japan. Patent No: EP 1437137 B1⁽²⁾Combination of both these drugs may help to improve outcomes after cerebral ischemia. Edaravone, a free radical scavenger, is a novel neuroprotective agent, and Argatroban is a selective thrombin inhibitor. Both the drugs were approved by the Japanese Government, and have frequently been used for the treatment of acute brain infarction in Japan.(clinical trials)⁽³⁾

A. Edaravone⁽^4-12)

Figure 1 : Chemical Structure of Edaravone

Edaravone is a neuroprotective agent used for the purpose of aiding neurological recovery following acute brain ischemia and subsequent cerebral infarction⁽⁴⁾.It acts as a potent antioxidant and strongly scavenges free radicals, protecting against oxidative stress and neuronal apoptosis.^(6-7)The preventive effect is due to suppression of reperfusion caused oxidative endothelium cell damage since edarevone was found to inhibit 15-HPETE (15-hydroxy peroxy icosatetraenoic acid) as well as cortical edema of rat brain induced by arachidonate micro injection.

Edaravone has been shown to attenuate methamphetamine- and 6-OHDA(Ortho Hydroxy Dopamine) induced dopaminergic neurotoxicity in the striatum and substantianigra, and does not affect methamphetamine-induced dopamine release or hyperthermia.^(8,9)It has also been demonstrated to protect against MPTP-mediated dopaminergic neurotoxicity to the substantianigra, though notably not to the striatum.^(10-12)

B. Argatroban⁽^13,14)

Figure 2: Chemical structure of Argatroban

Due to its selective inhibitory mechanism, argatroban blocks both circulating and clot-bound thrombin. Argatroban is a direct thrombin inhibitor that binds reversibly to the catalytic site of thrombin and that does not require other cofactors to exert its antithrombotic action. Argatroban exerts its anticoagulant effects by inhibiting thrombin-catalyzed or induced reactions, including fibrin formation; activation of coagulation factors V, VIII, and XIII; activation of protein C; and platelet aggregation.

A rapid onset of its anticoagulant action is achieved after intravenous administration. The elimination half-life of argatroban is (52+/-16 minutes) which is short enough to ensures a rapid restoration of hemostasis upon cessation of treatment. Argatroban produces a predictable dose response, and its anticoagulant actions can be monitored easily through the routine coagulation tests activated partial thromboplastin time (aPTT) and activated clotting time (ACT).

The review of literature regarding quantitative analysis of Edaravone and Argatroban revealed that no attempt was made to develop analytical methods for Edaravone and Argatroban. Some spectrometric methods and chromatographic methods have been reported for the estimation of the individual drugs.⁽^15-29)

MATERIAL AND METHODS:

Apparatus and instrument:

Ø A double beam UV/Visible spectrophotometer (Shimadzu model 2450, Japan) with spectral width of 2 nm, 1 cm quartz cells was used to measure absorbance of all the solutions.

Ø Spectra were automatically obtained by UV-Probe system software.

Ø An analytical balance (Sartorius CD2250, Gottingen, Germany) was used for weighing the samples.

Ø Sonicator(D120/2H, TRANS-O-SONIC)

Ø Class ‘B’ volumetric glassware were used (Borosillicte)

Ø All instruments and glass wares were calibrated.

Reagents and material:

Ø Edaravone (Gift sample from BDR Pharmaceutical International Pvt. Ltd.)

Ø Argatroban (Gift sample from BDR Pharmaceutical International Pvt. Ltd.)

Ø Methanol AR Grade (FINAR), Distilled Water, NaOH AR Grade (RANCHEM), HCl (ASTRON) was used for development purpose.

First derivative conditions:

Ø Mode : Spectrum

Ø Scan speed : Fast

Ø Wavelength range : 200-400 nm

Ø Derivative order : first

Ø Scaling factor: 1

Preparation of standard solutions:

Standard solution of Edaravone (EDA):

Preparation of stock solution of EDA:

Accurately weighed quantity of EDA 10 mg was transferred to 100ml volumetric flask, dissolved, and diluted up to mark with methanol to give a stock solution having strength 100µg/ml.

Standard solution of Argatroban (ARG):

Preparation of stock solution of ARG:

Accurately weighed quantity of ARG 10 mg was transferred into 100 ml volumetric flask, dissolved and diluted up to mark with methanol to give a stock solution having strength 100µg/ml.

Preparation of Standard Mixture Solution (EDA + ARG):

1ml of standard stock solution of EDA (100μg/ml) and 1ml of standard stock solution of ARG (100μg/ml) were pipetted out into two 10ml volumetric flasks and volume was adjusted to the mark with methanol to get 10μg/ml of EDA and 10μg/ml of ARG.

Preparation of test solution:

The preparation of synthetic mixture was as per patent:

Ø Edaravone : 30mg

Ø Sodium Bisulphite : 30mg

Ø Argatroban:10mg

Ø Phosphoric acid: q.s (adjust pH4.5)

Ø Water for Injection : 7.5 ml (finally upto 25ml)

All the excipients were mixed in 100ml volumetric flask dissolved in 25 ml of distilled water and sonicated for 15min. make up the volume with methanol up to 100 ml. The solution was filtered through Whatman filter paper No. 42.

Finally the solution had concentration 300μg/ml for EDA and 100μg/ml for ARG.

Validation of proposed method:

Parameters to be considered for the validation of methods are:

1) Linearity and range

Procedure:

The linearity response was determined by analyzing 6 independent levels of calibration curve in the range of 10-35μg/ml and 10-35μg/ml for EDA and ARG respectively (n=6).

Calibration curves for EDA:

This series consisted of six concentrations of standard EDA solution ranging from 10-35μg/ml. The solutions were prepared by pipetting out Standard EDA stock solution (1ml, 1.5ml, 2ml, 2.5ml, 3ml, 3.5ml) was transferred into a series of 10 ml volumetric flask and volume was adjusted up to mark with Methanol. A zero order derivative spectrum of the resulting solution was recorded and processed to first derivative spectra, measured the absorbance at 280.47nm against a reagent blank solution (Methanol). Calibration curve was prepared by plotting absorbance versus respective concentration of EDA.

Calibration curve for ARG:

This series consisted of six concentrations of standard ARG solution ranging from 10-35μg/ml. The solutions were prepared by pipetting out Standard ARG stock solution (1ml, 1.5ml, 2ml, 2.5ml, 3ml, 3.5ml) was transferred into a series of 10 ml volumetric flask and volume was adjusted up to mark with Methanol. A zero order derivative spectrum of the resulting solution was recorded and processed to first derivative spectra, measured the absorbance at 351.0 nm against a reagent blank solution (Methanol). Calibration curve was prepared by plotting absorbance versus respective concentration of ARG.

2) Precision

I. Intraday precision

Procedure

Ø The precision of the developed method was assessed by analyzing samples of the same batch in nine determinations with three Standard solutions containing concentrations 10,15,20μg/ml for EDA and 10,15,20μg/ml for ARG and three replicate (n=3)each on same day.

Ø First-derivative absorbance (D₁) was measured at 280.47nm for EDA and 351.0nm for ARG.

Ø The % RSD value of the results corresponding to the absorbance was expressed for intra-day precision.

II. Interday Precision

Procedure

Ø First-derivative absorbance (D₁) was measured at 280.47nm for EDA and 351.0nm for ARG.

Ø The % RSD value of the results corresponding to the absorbance was expressed for inter-day precision.

3) Accuracy

It was determined by calculating the recovery of EDA and ARG by standard addition method.

Accuracy was done by adding both API standard solution and test solution. Total concentration was as per table 1

Procedure

Table 1: _{Solutions for Accuracy
Study}

Concentration of Formulation (µg/ml)		Concentration of API in spiking solution (µg/ml)		Total concentration of (μg/ml)
Concentration of Formulation (µg/ml)		Concentration of API in spiking solution (µg/ml)		EDA	ARG
EDA	ARG	EDA	ARG	EDA	ARG
15	5	12	4	27	9
15	5	15	5	30	10
15	5	18	6	33	11

Each solution was taken and diluted with Methanol up to 10ml volumetric flask and scanned between 200nm to 400nm against Methanol as a blank. The amount of EDA and ARG was calculated at each level and % recoveries were computed.

4) LOD (Limit of Detection) and LOQ (Limit of Quantification):

The Limit of detection and Limit of Quantification of the developed method was assessed by analyzing ten replicates of standard solutions containing concentrations 10μg/ml for EDA and 10μg/ml for ARG.

The LOD and LOQ may be calculated as

Where, SD = ten replicates of absorbance

Slope = the mean slope of the 6 calibration curves

5) Robustness and ruggedness

Robustness and Ruggedness of the method was determined by subjecting the method to slight change in the method condition, individually, the:

Ø Change in Stock Solution Preparation,

· Stock-1 (10mg EDA in 100ml Methanol -100 μg/ml and 10mg ARG in 100ml Methanol - 100 μg/ml)

· Stock-2 (10mg EDA in 50ml Methanol -200μg/ml and 10mg ARG in 50ml Methanol - 200 μg/ml)

Ø Change in instrument (UV-Vis Spectrophotometer model 1800 and 2450),

Three replicates were made for the concentration (10,15,20 μg/ml of EDA and 10,15,20 μg/ml of ARG) with different stock solution preparation and the recording of absorbances were done on both the UV-Vis spectrophotometer. % RSD was calculated.

Analysis of EDA and ARG in synthetic mixture:

Composition of synthetic mixture

The preparation of synthetic mixture was as per patent:

Ø Edaravone : 30mg

Ø Sodium Bisulphite : 30mg

Ø Argatroban:10mg

Ø Phosphoric acid: q.s (adjust pH4.5)

Ø Water for Injection : 7.5 ml (finally upto 25ml)

Ø All the excipients were mixed in 100ml volumetric flask dissolved in 25 ml of distilled water and sonicated for 15min. make up the volume with methanol up to 100 ml. The solution was filtered through Whatman filter paper No. 42.

Finally the solution had concentration 300μg/ml for EDA and 100μg/ml for ARG.

From that pipette out 1 ml in 10 ml volumetric flask and volume was made up to mark with methanol to obtain final solution containing 30µg/ml of EDA and 10µg/ml of ARG. A zero order derivative spectrum of the resulting solution was recorded and processed to first derivative spectra. A first order derivative spectrum of the sample solution was recorded and the absorbance at 280.47nm and 351.0nm were noted for estimation of EDA and ARG, respectively. The concentrations of EDA and ARG in formulation were determined using the corresponding calibration graph.

RESULT AND DISCUSSION:

Selection of wavelength and method development for determination of edaravone and argatroban

The standard solution of EDA and ARG were scanned separately between 200-400nm, and zero-order spectra were not showed overlapping peaks.

_{Thus obtained spectra were then processed to obtain first-derivative spectra.}

First order derivative spectrum for EDA showed zero crossing points: 345.16, 351.0, 356.33. The wavelength selected for estimation of EDA was 351.0nm because it showed r²>0.999 at this wavelength in mixture.

First order derivative spectrum for ARG showed Four zero crossing points: 212.96, 241.13, 259.15, 280.47nm. The wavelength selected for estimation of ARG was 280.47nm because it showed r²>0.999 at this wavelength in mixture (Figure 3)

_{Table
5:Recovery
data of EDA *(n=3)}

Conc. of EDA from formulation (µg/ml)	Amount of Std. EDA added (µg/ml)	Total amount of EDA (µg/ml)	Total amount of EDA found (µ*g/ml) Mean ± SD**	% Recovery (n=3)	% RSD EDA
15	0	15	15.17±0.000230	101.13%	0.598
15	12	27	27.3 ± 0.000200	101.11 %	0.738
15	15	30	30.2 ± 0.000264	100.66 %	0.877
15	18	33	33.6 ± 0.000208	101.81 %	0.617

_{Table
6: Recovery data
of ARG*(n=3)}

Conc. of ARG from formulation (µg/ml)	Amount of Std. ARG added (µg/ml)	Total amount of ARG(µg/ml)	Total amount of ARG found (µ*g/ml) Mean ± SD**	% Recovery (n=3)	% RSD ARG
5	0	5	4.99 ± 0.000057	99.80	0.212
5	4	9	9.00 ± 0.000050	100.00 %	0.158
5	5	10	10.75 ± 0.000030	100.75 %	0.141
5	6	11	11.20 ± 0.000028	101.81 %	0.636

_{Table
7: LOD and LOQ data of EDA and ARG
*(n=10)}

Conc. (μg/ml)		*Avg. ± SD (280.47 nm) EDA**	% RSD	*Avg. ± SD (351.0 nm) ARG**	% RSD
EDA	ARG
10	10	-0.0117 ± 0.000316	0.266	-0.0040 ± 0.000193	0.468
LOD (μg/ml)		1.04		1.59
LOQ (μg/ml)		3.16		4.82

_{Table 8: Robustness and Ruggedness
data of
EDA and ARG *(n=3)}

_{Conc. (μg/ml)}		_Edaravone_{(Mean* ±% RSD) (n=3)}
		_{Different Instrument}				_{Different Stock}
		_UV-2450		_UV-1800		_{Stock – 1}		_{Stock – 2}
₁₀		_{-0.0121 ± 0.826}		_{-0.0119 ± 0.924}		_{-0.0119 ± 0.924}		_{-0.0120 ± 0.375}
₁₅		_{-0.0160 ± 0.354}		_{-0.0159 ± 0.358}		_{-0.0164 ± 0.609}		_{-0.0170± 0.588}
	₂₀		_{-0.0210 ± 0.271}		_{-0.0211± 0.805}		_{-0.0219 ±0.260}		_{-0.0221 ± 0.261}
		_Argatroban_{(Mean ±% RSD) (n=3)}
₁₀		_{-0.0040 ± 0.142}		_{-0.0039 ± 0.146}		_{-0.0041 ± 0.139}		_{-0.0042 ± 0.261}
	₁₅		_{-0.0051 ± 0.215}		_{-0.0049 ± 0.142}		_{-0.00521±0.117}		_{-0.0059± 0.966}
	₂₀		_{-0.0080 ± 0.712}		_{-0.0079 ± 0.721}		_{-0.0081 ± 0.703}		_{-0.0078 ± 0.730}

_{Table
9: Analysis data of commercial formulation *(n=3)}

Sr. No.	Formulation (synthetic mixture)		*Absorbance (280.47nm) EDA**	% Assay EDA±SD	Absorbance* (351.0nm) ARG	% Assay ARG±SD
	EDA	ARG
1	30	10	-0.0300	100.00 ± 0.000115	-0.0039	99.00 ± 0.0000529
2			-0.0310		-0.0038
3			-0.0300		-0.0039

_{Table 10: Summary of validation parameters}

PARAMETERS	Absorbance correction method
PARAMETERS	Edaravone	Argatroban
Concentration range(µg/ml)	10-35	10-35
Regression equation	y = -0.001x + 0.001	y = -0.0004x +0.000
Correlation Coefficient(r²)	0.9980	0.9990
Accuracy(%Recovery) (n=3)	100.17	100.58
Intra-day Precision (%RSD) (n=3)	0.263 - 0.480 %	0.510 –0.989%
Inter-day precision (%RSD) (n=3)	0.256-0.476%	0.256-0.476%
LOD(µg/ml)	1.04µg/ml	1.59µg/ml
LOQ(µg/ml)	3.16µg/ ml	4.82 µg/ml
Ruggedness and Robustness(% RSD)	0.260- 0.924%	0.117 – 0.966%
% Assay	100.0	99.0

CONCLUSION:

All the parameters are validated as per ICH guidelines for the method validation and found to be suitable for routine quantitative analysis in pharmaceutical dosage forms. The result of linearity, accuracy, precision proved to be within limits with lower limits of detection and quantification. Ruggedness and Robustness of method was confirmed as no significant were observed on analysis by subjecting the method to slight change in the method condition. Assay results obtained by proposed method are in fair agreement.

ACKNOWLEDGEMENT:

We are sincerely thankful to Shree Dhanvantary Pharmacy College, Kim, Surat, for providing us Infrastructure facilities and moral support to carry out this research work. We are also thankful to SDPARC for giving us their special time and guidance for this research work. We also thank our colleagues for their helping hand.

REFERENCES:

1. Harukuni I and Bhardwaj A. Mechanisms of brain injury after global cerebral ischemia. Neurologic Clinics. 24(1) ; 2006 : 1–21.

2. Keiichi Tesseikai, Katsumi C/o. Drugs comprising combination of anti thrombotic agent with pyrazolone derivative. European Patent EP1437137B1, 2008.

3. Combination therapy for Acute ischemic stroke study group. Edaravone and Argatroban stroke therapy study for acute ischemic stroke. 2008, Available from:http://www. ClinicalTrials.gov./show/NCT00153946.

4. Doherty AM, Annual reports in Medicinal Chemistry, Boston: Academic Press, 2002, pp 37.

5. Watanabe T. et. al. Research and development of the free radical scavenger Edaravone as a neuroprotectant. Yakugaku Zasshi (in Japanese), , 124(3); 2004: 99–111.

6. Higashi Y, Jitsuiki D, Chayama K and Yoshizumi M, Edaravone (3-methyl-1-phenyl-2-pyrazolin-5-one). a novel free radical scavenger, for treatment of cardiovascular diseases. Recent Patents on Cardivascular Drug Disorder. 1(1); 2006: 85–93.

7. Yoshida H. et. al. Neuroprotective Effects of Edaravone: A novel free radical scavenger in cerebrovascular injury. CNS Drug Review, 12(1); 2006: 9–20.

8. Yuan WJ et. al. Neuroprotective effects of Edaravone-administration on 6-OHDA-treated dopaminergic neurons. BMC NeuroScience. 9(1); 2008: 75.

9. Kawasaki T et. al. Protective effect of the radical scavenger Edaravone against methamphetamine-induced dopaminergic neurotoxicity in mouse striatum. European Journal of Pharmacology. 542(1-3); 2006: 92–99.

10. Kawasaki T et.al. Edaravone (3-methyl-1-phenyl-2-pyrazolin-5-one), a radical scavenger, prevents 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine Induced Neurotoxicity in the Substantianigra but not the Striatum. Journal of Pharmacology and Experimental Therapeutics. 322(1); 2007: 274–281.

11. Yokoyama H et. al., Role of Reactive nitrogen and reactive oxygen species against MPTP neurotoxicity in mice. Journal of Neural Transmission, 115(6); 2008: 831–842.

12. Yokoyama H et. al. Comparative pharmacological study of free radical scavenger, nitric oxide synthase inhibitor, nitric oxide synthase activator and cyclooxygenase inhibitor against MPTP neurotoxicity in mice. Metabolic Brain Disorder, 23(3); 2008: 335–349.

13. Escolar G, Bozzo J and Maragall S. Argatroban a direct thrombin inhibitor with reliable and predictable anticoagulant action. Drug today. 42(4); 2006: 223-236.

14. Drug Profile, Available from: www.chemicalbook.com/ProductMSDSDetailCB1287462_EN.htm

15. Ping Liao, Zheng-Yu Yan and Xiao Sun. A novel fluorescent assay for Edaravone with aqueous functional CdSe quantum dots. Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy. 72(5); 2009: 1066-1070.

16. Gandhimathi M, Kumar MS, Baghla R and Ravi TK. RP-HPTLC Method for the In Vitro Estimation of Edaravone in Human Plasma. Indian Journal of Pharmaceutical science.72(2); 2010: 276-282.

17. Li jin-lin et. al. Determination of Phenylhydrazine Residues in Edaravone by HPLC. Institute of Medical Information, CAMS. 2(1); 2009: 24-28.

18. Fu Gui-Ying, WEN Ming-Ling and ZUO Xiu Ping. Determination of content and related substance of Edaravone injection by HPLC. Journal of Chinese people´s Liberation Army. 2(1); 2009: 101-14.

19. WEI Min, XIAO Yi. Determination of concentration of Edaravone in human serum by RP HPLC. Clinical pharmacology. 10(2); 2007-08: 142-143.

20. Patel Bhumi K et. al. Method development and validation of RP HPLC for simultaneous estimation of Edaravone and Citicoline sodium in synthetic mixture. inventi rapid: pharmaceutical analysis and Quality Assurance publication.1(1); 2014: 31-35.

21. Tang DQ, Bian TT and Jiang SS, LC-MS/MS methods for the determination of Edaravone and/or Taurine in rat plasma and its application to a pharmacokinetic study. Biomed Chromatography. 28(9); 2014: 1173-82.

22. Patel Bhumi K, Raj Hasumati A and Jain Vineet C, Simultaneous Estimation of Edaravone and Citicoline sodium by Ratio derivative spectroscopic method in synthetic mixture. Pharmascience Monitor, An International Journal of Pharmaceutical Scence. 5(2); 2014: 118-128.

23. Patel Bhumi K, Raj Hasumati A, Jain Vineet C, First Derivative Spectroscopic Method For Simultaneous Estimation of Edaravone and Citicoline Sodium in Synthetic Mixture. Research and Reviews, Journal of Pharmaceutical Analysis. 3(2); 2014: 37-44.

24. Singh Atul Pratap, Eswari, T. S., Gurusharan, and Verma V. Formulation and Evaluation of Parenteral Drug Edaravone. International Journal of Pharmaceutical Research Scholars, , 3(4); 2014: 134-141.

25. Supplement to Japanese Pharmacopoeia, The Ministry of Health, Labour And Welfare, 15^thEdn; Official From October 1, 2009, pp 2087, 2088.

26. Ahmad S, Ahsan A and George M, Simultaneous monitoring of Argatroban and its major metabolite using an HPLC method: potential clinical application. Clinical and applied thrombosis-hemostasis. 5(4); 1999: 252-8.

27. GUO Xu-guang, ZHENG Zi-dong, Content Determination of Argatroban Injection by RP-HPLC. China Phamacy. 2013: 33.

28. GUO Da-qing, et al., Methodology on determination of Argatroban in human plasma. Central South Pharmacy, , 3(1); 2009: 134-137.

29. Rhea JM, Snyder ML and Winkler AM, Development of a fast and simple liquid chromatography-tandem mass spectrometry method for the quantitation of Argatroban in patient plasma samples, Journal of chromatography. B, Analytical technologies in the biomedical and life sciences. 15, 2012; 893-894:168-72.

30. Beckett AH, Stenlake JB, Davidson AG. Ultraviolet-Visible absorption spectrophotometry, Practical Pharmaceutical Chemistry, 4th Edn; New Delhi, CBS Publishers, 2002, pp 275-300.

31. International Conference on Harmonization, Harmonized Tripartite Guideline, Validation of Analytical Procedures Text and Methodology, ICH Q2 (R1), 2005.

Received on 02.02.2015 Accepted on 28.02.2015

Asian J. Res. Pharm. Sci. 5(1): Jan.-March 2015; Page 27-35

DOI: 10.5958/2231-5659.2015.00005.3

Sr. No	Concentration (μg/ml)		*Absorbance (280.47nm)±SD EDA**	*Absorbance (351.0nm)±SD ARG**
Sr. No	EDA	ARG	*Absorbance (280.47nm)±SD EDA**	*Absorbance (351.0nm)±SD ARG**
1	10	10	-0.0118 ± 0.0007	-0.0038 ± 0.0007
2	15	15	-0.0159 ± 0.0012	-0.0058 ± 0.0011
3	20	20	-0.0210± 0.0013	-0.0080 ± 0.0006
4	25	25	-0.0254± 0.0010	-0.0101 ± 0.0007
5	30	30	-0.0312 ± 0.0013	-0.0121 ± 0.0007
6	35	35	-0.0360± 0.0015	-0.0140 ± 0.0006

Conc. (μg/ml)		*Abs. (EDA) Avg. ± SD (280.47nm)**	% RSD	*Abs. (ARG) Avg.± SD (351.0nm)**	% RSD
EDA	ARG	*Abs. (EDA) Avg. ± SD (280.47nm)**	% RSD	*Abs. (ARG) Avg.± SD (351.0nm)**	% RSD
10	10	-0.0120 ± 0.000057	0.480	-0.0038 ± 0.000057	0.510
15	15	-0.0158 ± 0.000064	0.405	-0.0058± 0.000057	0.989
20	20	-0.0210 ± 0.000057	0.263	-0.0080 ± 0.000063	0.781

Conc. (μg/ml)		*Abs. (EDA) Avg. ± SD(280.47nm)**	% RSD	*Abs. (ARG) Avg.± SD(351.0nm)**	% RSD
EDA	ARG	*Abs. (EDA) Avg. ± SD(280.47nm)**	% RSD	*Abs. (ARG) Avg.± SD(351.0nm)**	% RSD
10	10	-0.0121 ± 0.000057	0.476	-0.0048 ± 0.000028	0.583
15	15	-0.0162 ± 0.000057	0.351	-0.0060 ± 0.000057	0.950
20	20	-0.0222 ± 0.000057	0.256	-0.0081 ± 0.000055	0.679