INTRODUCTION:

Chemotherapy is usually systemic treatment of cancer. Regardless of the fact that chemotherapy improves survival, but it has its own toxicity and side effects, which comprise a negative impact on the patients’ quality of life. Though, Nausea and vomiting continue to be significant side-effects of cancer therapy and can affect patient compliance.¹ Therefore, it is important to provide prophylactic treatment for chemotherapy-induced nausea and vomiting (CINV). Thus, by addition of a neurokinin-1 receptor antagonist (NK1RA) (Aprepitant) to a standard 5-hydroxytryptamine (5-HT3) receptor antagonist (Ondansetron) and dexamethasone antiemetic regimen can be significantly improved the prevention of CINV throughout the overall period of risk (0-120 hours) of chemotherapy².

Dexamethasone (DEX) (Fig. 1a), is chemically 9-Fluoro-11b, 17, 21- trihydroxy-16a-methyl pregna-1, 4-diene-3, 20-dione a potent synthetic corticosteroid³. It is frequently used as an anti-inflammatory agent and also having immune suppressive properties. It is official in Indian Pharmacopoeia⁴.Similarly, Ondansetron (Fig.1b) is chemically 9(2S, 5R, 6R)-6- [3-(2, 6-dichlorophenyl)-5-methyl-1, 2-oxazole-4-amido]-3,3-dimethyl-7-oxo-4-thia-1 aza bi-cyclo [3.2.0] heptane-2-carboxylic acid12 ⁵.It is penicillinase resistant penicillin used in the treatment of bacterial infections such as pneumonia, skin and urinary tract infection⁶. It is official in IP⁷. Likewise, Aprepitant (APT) (Fig.1c) is chemically 5-[[(2R, 3S)-2-[(1R)-1-[3, 5-bis-(trifluoromethyl) phenyl] ethoxy]-3-(4-fluorophenyl)-4-morpholinyl] methyl]-1, 2dihydro-3H-1, 2, 4-triazol-3-one. It is a selective high affinity antagonist of human substance P/neurokinin 1 (NK1) receptors. It is having little or no affinity for serotonin (5-HT3), dopamine, and corticosteroid receptors⁸. Currently, none of the information is available concerning the fixed dose combination of these selected drugs. Although, these three drugs can be administrated simultaneous to prevent acute and delayed CINV before chemotherapy. The recommended dose for CINV includes day 1, APT 125 mg, ondansetron (OND) 8 mg, and dexamethasone 12 mg before chemotherapy and OND 8 mg 8 hours later; days 2 through 3, APT 80mg qd⁹. Therefore, there is need to establish analytical methods for the estimation of these three drugs. Literature survey reveals the presence of numerous methods for the determination of these three drugs in different matrices. A couple of published review articles where they provided information about the few analytical methods such as HPLC methods and spectrophotometric methods^10,11available for OND, APT and DEX in the individual and combinations with other drugs. To the best of our knowledge one HPLC and UV-PLS method in laboratory prepared organo-gel formulation of these three antiemetic drugs has been recently published.¹² The fact of that till now no HPTLC method has been reported for simultaneous estimation of these three selected drugs. Hence, this encourages us to investigate the development of HPTLC procedure, which may applicable for the routine quality control analysis of their laboratory prepared mixtures.

Fig. 1 Chemical structure of a) Ondansetron b) Aprepitant and c) Dexamethasone

Nowadays, HPTLC is widely used due to low maintenance cost, low solvent consumption per sample and need for minimum sample cleanup and having automatic sample applicator and scanner. Therefore, it is enough to analyze different kinds of samples simultaneously. Due to these advantages, it gives more precise, accurate, sensitive and robust results. In spite of that development of HPTLC method is a tedious procedure that requires instantaneous determination of several factors. Therefore, the effect of simultaneously varying several factors on the responses of developed method was optimized by applying experiment of design¹³. In this study, a central composite design (CCD) along with response surface methodology was mainly chosen due to its flexibility, in terms of experimental runs and information related to factor's main and interaction effects. Therefore, HPTLC method was developed and validated (ICH 2005) for simultaneous estimation of OND, APT and DEX in laboratory prepared ternary mixtures, and optimized by using CCD design and response surface methodology for robustness testing.

MATERIALS AND METHODS:

Instruments

Precoated silica gel 60F254 aluminum plates (10×10 cm, 100μm thickness; Merck, Darmstadt, Germany), Micro syringe (Linomat syringe 659.0014, Hamilton Bonaduz Schweiz, Camag, Switzerland), Linomat V applicator (Camag, Switzerland), Twin trough chamber (10×10 cm; Camag, Switzerland), Hair dryer (Panasonic), UV chamber (Camag, Switzerland) and TLC scanner III (Camag, Switzerland), WinCATS version 1.4.6 software (Camag, Switzerland) was used in the study. All data analysis of experimental design was performed by using the Design Expert trial version 10.0.0 (Stat Ease Inc., Minneapolis, USA).

Chemicals and Reagents

Analytically pure APT (Chandra Labs, Kukatpally, India), OND (Hetero Drugs Ltd, Hyderabad, India) and DEX (Cadila Ltd. Ahmadabad, India) were obtained as gift sample (99.7% - 99.9% purity). All solvents used were of analytical grade and purchased from Merck Specialties Pvt. Ltd., Mumbai, India.

Sample preparation

Preparation of standard solutions of OND, APT and DEX

10 mg of each drug OND, APT and DEX were weighed accurately and stock solutions were prepared separately followed by dilution with methanol in 10 mL volumetric flask to obtain a concentration of 1000 μg/mL. Aliquots of stock solutions were appropriately transferred and diluted with methanol in 10 mL volumetric flask to prepare a final concentration of 100μg/mL for OND, APT and DEX.

Preparation of laboratory prepared ternary mixtures of OND, APT and DEX

From the aforementioned standard stock solutions, mixed standard solution was prepared by dissolving appropriate concentration of the stocks in methanol and used for the estimation of individual drugs from the ternary mixtures.

Application of the HPTLC method

Prewashing and Activation of pre-coated plates

Precoated silica gel plates exposed to high humidity or kept on hand for long time requires prewashing and activation. Plate was prewashed with methanol and activated by placing them in an oven at 110-120ºc for 30 min prior to spotting.

Chromatographic development and scanning

Standard solutions of different concentrations were spotted with a micro-syringe in the form of bands (band width of 6 mm) on Activated pre-coated silica gel aluminum Plate 60 F254using a Camag Linomat V sample applicator. Linear ascending development was carried out in a twin trough glass chamber. The mobile phase consisted of chloroform: methanol: acetone: ethyl acetate: ammonia (9:4:2:5:0.2 % v/v/v/v/v). Mobile phase was thoroughly mixed and poured in twin trough chamber and left the chamber for saturation before each run with the filter paper for 30 min. The length of the chromatographic run was 8 cm. TLC plates were dried with the help of hair dryer. Densitometric scanning was performed by using Camag TLC scanner III with winCATS software (V 1.4.6.2002, Camag). All measurements were taken at 254 nm in the absorbance mode, slit dimension (6.0×0.30 mm, micro), scanning speed 20 mm/s, data resolution 100 μm/step, optical filter (second order), filter factor (Savitsky golay 7). The deuterium lamp was used as source of radiation and emitting a continuous UV spectrum in the range of 190-400 nm. Concentration of the drug was obtained from the intensities of diffusely reflected lights. Evaluation was performed by peak areas with linear regression analysis.

Software assisted Method optimization

Central composite design, as a three-level factorial design with k factors, requires 2 factorial runs, 2k axial runs symmetrically spaced at along each variable axis and at least one centre point. The factors and ranges preferred for consideration were based on previous univariate studies of method development and chromatographic observation. Twelve experiments with two centre points were performed by selection of three factors, developing distance (A), acetone content in mobile phase (B), Chamber saturation time (C) and retention factor (Rf) of OND, DEX and APT were the responses of these three drugs, as shown in Table 1. The nominal value for all these three factors, A, B and C, were 8 cm, 2 mL and 30 min, respectively. In perspective to this, developing distance (A) was kept between 6.59 and 9.41 cm. Similarly, minimum and maximum content of Acetone (B) were fixed as 1.9 and 2.1 mL, respectively. Likewise, minimum and maximum values for saturation time (C) were selected as 22.93 and 37.07 min, respectively. The coded value of α is 1.41. The data generated were analyzed using Design Expert (Version 10.0.0, Stat Ease Inc., Minneapolis, USA) statistical software. The significance of the significant factors was designed using Fisher's statistical test for Analysis of Variance (ANOVA) model that were estimated. ANOVA for linear regression partitions the total variation of a sample into components. These components were then used to compute an F-ratio that evaluates the effectiveness of the model. If the probability associated with the F-ratio is low, the model is significantly considered a better statistical fit for the data¹⁴. All experiments were performed in randomized order to minimize the bias effects of uncontrolled factors.

Table 1: Values of variables at factorial, axial and centre points of the experimental design (CCD)

Run	Type	Factors			Response R_f
Run	Type	Developing distance (cm)	Acetone content in mobile phase (ml %)	Saturation time (mm)	APT	OND	DEX
1	Axial	1.41	0	0	0.81	0.54	0.28
2	Axial	0	0	1.41	0.80	0.56	0.26
3	Centre	0	0	0	0.77	0.53	0.23
4	Centre	0	0	0	0.77	0.53	0.23
5	Axial	0	1.41	0	0.76	0.57	0.24
6	Factorial	1	-1	1	0.8	0.51	0.27
7	Axial	0	0	-1.41	0.75	0.53	0.22
8	Axial	0	-1.41	0	0.76	0.5	0.24
9	Factorial	-1	-1	-1	0.74	0.51	0.23
10	Factorial	1	1	-1	0.76	0.55	0.22
11	Axial	-1.41	0	0	0.73	0.52	0.23
12	Factorial	-1	1	1	0.73	0.55	0.22

n=3 replicates, APT: Aprepitant, CCD: central composite design, DEX: Dexamethasone, OND: Ondansetron

METHOD VALIDATION:

Linearity and Range

A stock solution containing 100μg/mL of each drug OND, APT and DEX were prepared in methanol. Different volumes of this solution were applied to the plate resulting in application of 200-1200ng/band for OND, 500-1000ng/band for APT and 1000-2000ng/band for DEX. Each concentration was applied on the developed plate as described above. Peak areas were plotted against corresponding concentrations to obtain the calibration plot.

Precision and Accuracy

Precision of the developed method was evaluated by performing repeatability on same day and intermediate precision studies on different days in three replicates. Repeatability and intermediate precision were carried out for three different concentration (200, 600 and 1000 ng/band for OND, 600, 800 and 1000 ng/band for APT and 1000, 1400 and 1800 ng/band for DEX). Peak area was measured and expressed in terms of percent relative standard deviation (%RSD).

The accuracy of the method was determined in triplicate at three concentration levels of 80%, 100% and 120% by spiking the prequantified samples with a known amount of OND, APT and DEX standard. Recovery studied was calculating in term of % RSD for aforementioned three drugs. The good recoveries of standard addition method suggested good accuracy of the proposed methods.

Sensitivity

As per ICH guideline, limit of detection (LOD) and limit of quantitation (LOQ) of the developed method were calculated from the standard deviation of the response and slope of the calibration curve of each drug using the formulae, limit of detection (3.3×σ/S) and limit of quantitation (10×σ/S), where, σ is standard deviation of response and S is the slope of calibration curve.

Selectivity and Specificity

The selectivity of the proposed method for the simultaneous determination of the cited drugs was performed through the analysis of laboratory prepared ternary mixtures. These were prepared in order to contain a combination of the three drugs at different ratios within their linearity ranges. The specificity of the method was ascertained by comparing the R value and spectra of standard drug and laboratory prepared ternary mixtures of OND, APT and DEX. The peak purity of each drug was assessed by comparing the spectra at three different levels, i.e., peak start (S), peak apex (M) and peak end (E) position.

Robustness

The effect of small and deliberate changes in the method parameter, such as a change in the distance travelled, acetone content in the mobile phase by volume, chamber saturation time and wavelength were evaluated. The effect of these changes on all the Rf values and peak areas were examined by calculating the % RSD for each parameter.

Analysis of Laboratory prepared ternary mixtures of OND, APT and DEX

Different mixtures of each drug were prepared by transferring different volumes of OND, APT and DEX from working solutions into 10 mL volumetric flasks and diluted with methanol. From these ternary mixture solutions, 5-μL portions were spotted on HPTLC plates to obtain final concentrations within the specified linearity ranges (Table 4). Spot application, plate development and scanning were performed as mentioned under Instrumentation. The peak area of each drug in every mixture was recorded and the recovered concentration was calculated from the corresponding regression equation.

RESULTS AND DISCUSSIONS:

Method optimization

The chromatographic conditions were optimized in order to develop an HPTLC method for the simultaneous measurement of laboratory prepared ternary mixtures of OND, APT and DEX.

Preliminary study for selection of mobile phase

Preliminary trials were performed on 10 cm TLC Plate, for the separation of all three cited drugs. Mobile phase for this method was selected on the basis of analysis of own experience, literature report of similar studies and traditional trial and error methods. Though, various combination of solvent of different polarities such as methanol, ethyl acetate, acetone, chloroform, toluene, acetonitril, glacial acetic acid and ammonia were tried in different ratio to resolve the peak of OND, APT and DEX. Initially Mobile phases composed of [ethyl acetate and ethanol], [ethyl acetate and methanol], [chloroform and methanol], [toluene and methanol], [toluene and ethanol] or [chloroform and acetone] in different ratio were tried, but failed to resolve the investigated drugs. Although the mobile phase containing [chloroform: methanol: ethyl acetate in 5:5:5 %v/v/v] gave slightly better resolutions, but co-elution of DEX and APT was observed. Further, changing the ratio of mobile phase [chloroform: methanol: ethyl acetate in 9:4:5%v/v/v] OND peak was observed with the solvent front. Therefore, the acetone was added in above mentioned mobile phase and the diffuse spot of OND was observed. Gradual addition of acetone with different ratio to the above mobile phase [chloroform: methanol: ethyl acetate and acetone in 9:4:5:1, 9:4:5:1.5 and 9:4:5:2%v/v/v/v] resulted in a significant improvement in resolution but tailing was observed. Therefore, ammonia seemed as an appropriate additive to this mobile phase in order to overcome spot tailing as well as to enhance peak shape and resolution. Ultimately mobile phase consisted of chloroform: methanol: acetone: ethyl acetate: ammonia (9:4:2:5:0.2 %v/v/v/v) were studied at saturation time of 30 min and solvent migration distance of 8 cm. Rf values of OND, APT and DEX were found to be 0.54± 0.02, 0.79±0.02 and 0.23±0.01, respectively. The optimized procedure resulted in well-defined spots with reproducible Rf values (Fig.4) when plate was scanned at 254 nm.

Optimization of chromatographic conditions using CCD

CCD approach was selected due to its flexibility and applied to optimize the HPTLC separation by achieving a better understanding of the factor’s main and interaction effects [34-38]. A three-factorial, rotatable central composite statistical experimental design was employed using 12 experimental runs that included three centre points. The independent variables, such as the developing distance (A), acetone content in mobile phase (B), saturation time (C) and the responses (Rf values) for all 12 optimized trial experimental runs were tabulated in Table 1. During model selection, the best-fitted models for the Rf values of OND, DEX and APT were a linear model, based on the lowest Prediction Residual Error Sum of Squares (PRESS) value and adjusted R2 value closer to 1. The model was validated with an analysis of variance (ANOVA) using the Design Expert software and the results were shown in Table 2. Significant effects had a P value less than 0.05. An adequate precision, a measure of the signal (response) to noise ratio, greater than 4 is desirable, and the obtained ratio for all three drugs indicated an adequate signal. A coefficient of variation (% CV) was less than 10% which measures the reproducibility of the model. The adjusted R2 values were high, it was indicated a good relationship between the experimental data and those of the fitted models, and the adjusted R2 values were within the acceptable limit of R2≥ 0.50, which indicated that the experimental data was fitted with polynomial equations (Dinç-Zor et al. 2020). The polynomial equation in terms of the actual components and factors was shown in Table 2. A positive value represents an effect that favors optimization, whereas a negative value indicates an inverse relationship between the factor and the response (Harang et al. 2001). Fig.2 and Fig.3 presented the Perturbation plots and 3-D response surface plots. It was constructed to evaluate the effect of the factors on the retention factor of each drug and also used for the predicted model to better understand the investigated procedure (Vladimir et al. 2002). This figure has shown that how the response changes in effect to perturbations in each factor from its defined reference value while all other factors are held constant at a reference point. Fig. 2 a) demonstrated that the acetone content in mobile phase (factor B) had the most significant effect on the Rf value of OND compared with other factors. Moreover, the developing distance (A) and chamber saturation time (C) had more significant effects on the Rf value of APT and DEX respectively, followed by the acetone content in mobile phase (factor B) as shown in Fig. 2 b) and Fig.2 c). Fig. 3 a) represented the variation in the retention factor of OND as a function of Acetone content in mobile phase and developing distance, while the chamber saturation time was kept constant. Likewise, Fig.3 b) and Fig.3 c) represented a variation in the Rf value of APT and DEX as a function of the saturation chamber time and developing distance while the acetone content in mobile phase was kept constant. An analysis of the perturbation plots and response surface plots of the optimization model revealed that the acetone content in mobile phase (B) and chamber saturation time (C) was more significantly affected factors. The results of optimized independent variables such as the developing distance (A), acetone content in mobile phase (B) and saturation time (C) were validated by comparing the predicted results and observed results.

Table 5: Intraday and Interday precision for ternary mixtures of OND, APT and DEX

Drug	Amount (ng/band)	Intraday precision (n=3)				Interday precision (n=3)
Drug	Amount (ng/band)	Mean area	S.D^a.	%RSD^b	S.E^c.	Mean area	S.D^a.	%RSD^b	S.E^c.
OND	200	733.8	6.57	0.89	2.68	754.5	10.70	1.41	4.37
	600	1731.3	5.71	0.33	2.33	1745.9	26.79	1.53	10.93
	1000	2857.1	5.34	0.19	2.18	2864.7	15.97	0.55	6.52
APT	600	1243.8	3.61	0.29	1.47	1262.4	18.69	1.48	7.63
	800	2128.5	5.92	0.27	2.41	2130.6	10.26	0.48	4.19
	1000	3103.1	5.89	0.19	2.39	3105.3	9.27	0.29	3.78
DEX	1000	1653.1	1.93	0.12	0.79	1662.1	6.88	1.01	6.89
	1400	2543.6	3.43	0.13	0.85	2549.8	22.52	0.88	9.19
	1800	3309.2	5.38	0.16	2.19	3317.4	1.08	0.52	6.97

n=3 Replicates; APT: Aprepitant, DEX: Dexamethasone, OND: Ondansetron, S.D^a=Standard Deviation; R.S.D^b=Relative standard deviation; S. E^c=Standard error

Limits of detection and quantification

LOD were found to be 23.07, 39.89 and 92.55ng/band for OND, APT and DEX respectively. Similarly, LOQ were found to be 71.12, 119.67 and 277.67ng/band for OND, APT and DEX respectively and shown in Table 4. The results were indicated that the methods have sufficient sensitivity.

Precision and Accuracy

The experiment was repeated three times in one day (intra-day precision) with different time interval. The average % RSD values and Standard error values for the peak area of OND, APT and DEX were found within range of 0.12-0.89% and 0.79-2.68 respectively. Similarly, the experiment was repeated on three different days (inter-day precision). The average % RSD values and standard error for the peak areas of OND, APT and DEX were found in range of 0.29-1.53% and 3.78-10.93, respectively. The results were shown in Table 5 and confirmed the good precision of the method.

The accuracy study has been performed by the standard addition method at three concentration level 80%, 100% and 120% by spiking with standard. The percentage recovery at these three levels were found in the range of 99.29-100.47% and percentage relative standard deviation (RSD %) values were found to be less than 2% in all cases. The results were shown in Table 6 and confirmed the accuracy and reliability of the developed method.

Specificity and Selectivity

The specificity of the HPTLC method for analysis of OND, APT and DEX was performed. The complete and clear separation was observed without any interference in retention factor as shown in Fig. 4.

Fig.4 HPTLC densitogram under optimized conditions showing Rf values of 0.23 for Dexamethasone (1000 ng/band), 0.54 for Ondansetron (200ng/band) and 0.79 for Aprepitant (500 ng/band).

The peak purity of analyzed drugs OND, APT and DEX was assessed by comparing their respective spectra at peak start, apex and end positions of the peak as shown in Fig. 5 (a-c) respectively. A good correlation (r value more than 0.999) was obtained for all drugs. Acceptable peak purity and correlation values suggested that there was no interference in the quantification of the three analyzed drugs in laboratory prepared ternary mixtures. This proven that the method is specific. Selectivity of the method was further examined by preparing several laboratory-prepared mixtures of the aforementioned drugs at various concentrations within the linearity ranges as mentioned in Table 4. The percentage relative standard deviation (RSD %) were found to be less than 2% and percentage relative standard error (%S.E.) were found within the range of 0.11-0.34% for OND, APT and DEX. The results were shown in Table 7 and satisfactory results were obtained that validating the selectivity of the methods.

Fig. 5 overlain peak purity spectra of a) Ondansetron b) Aprepitant and c) Dexamethasone with corresponding standard

Table 6

Recovery study of the method (using the standard addition method) for OND, APT and DEX

Drug	Initial amount (ng/band)	%Recovery level	Amount added (ng/band)	% Recovery	S.D^a.	%R.S.D^b
		50	100	99.29	0.51	0.52
OND	200	100	200	100.46	0.43	0.43
		150	300	100.41	0.91	0.90
		50	100	99.93	0.85	0.86
APT	200	100	200	100.18	0.15	0.16
		150	300	99.73	0.36	0.37
		50	250	99.60	0.56	0.56
DEX	500	100	500	100.03	0.14	0.14
		150	750	100.47	0.84	0.85

n=3 replicates, APT: Aprepitant, DEX: Dexamethasone, OND: Ondansetron, S.D^a.=Standard Deviation; R.S.D^b.=Relative standard deviation.

Table 7: Determination of OND, APT and DEX in laboratory-prepared mixtures by the proposed HPTLC method

Mixture	Nominal amount (ng/band)			Found (ng/band) (Mean ±S.D^a.)			%R.S.D^b			E^r(%)^c
Mixture	OND	APT	DEX	OND	APT	DEX	OND	APT	DEX	OND	APT	DEX
1	200	500	1000	201.02± 1.79	499.16± 1.72	1002.40± 3.14	0.89	0.35	0.31	0.34	0.14	0.12
2	800	600	1000	799.48± 2.28	601.16± 2.99	1001.16± 2.92	0.29	0.50	0.29	0.11	0.21	0.12
3	600	800	1000	600.16± 2.04	799.16± 3.06	1001.81± 4.08	0.34	0.38	0.41	0.14	0.16	0.17
4	1000	1000	1200	999.83± 2.56	998.66± 2.65	1198.15± 3.80	0.26	0.27	0.32	0.11	0.12	0.13
5	1000	800	1200	999.41± 3.55	798.16± 3.31	1197.31± 3.42	0.36	0.41	0.29	0.15	0.17	0.11

n=6 Replicate; APT: Aprepitant, DEX: Dexamethasone, OND: Ondansetron, S.D.^a= Standard deviation; RSD^b=Relative standard deviation; E^r(%)^c= Relative standard error

Robustness

For demonstrating the robustness of the method, slight variations in the optimized conditions were done, such as the Acetone content in the mobile phase, developing distance and chamber saturation time. The obtained results of %Relative standard deviations of the peak area were found to be less than 2% for all cited drugs. The results were shown in Table 8 and indicated the robustness of the method.

CONCLUSION

The sensitivity of the Rf values of OND, APT and DEX at various chromatographic variables such as acetone content in mobile phase, chamber saturation time and developing distance were concurrently optimized by using experimental design tool, response surface methodology and Derringer’s desirability function. The obtained results showed that the CCD approach is a flexible procedure that can reduce the number of necessary experiments for the development and optimization of an HPTLC method. In addition, it is an economic method that can be used to create utmost amount of information in less time by reducing the number of experiments. The optimized HPTLC method is simple, reliable and suitable for the rapid quantitative analysis of OND, APT and DEX in routine tests. Hence, the proposed HPTLC method can be successfully utilized for simultaneous estimation of OND, APT and DEX in laboratory prepared ternary mixture without interference and in pharmaceutical dosage form of individual drugs.

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Received on 14.06.2020 Modified on 19.07.2020

Research J. Pharm. and Tech 2021; 14(10):5531-5539.

DOI: 10.52711/0974-360X.2021.00965

Response (Rf value)	Type of model	Polynomial equation model for y	Adjusted R²	Model p value	%CV	Adequate precision
APT	Linear	0.77+0.025A-0.00626B+0.013C	0.7974	0.0011	1.58	11.51
OND	Linear	0.53+0.00354A+0.022B+0.0053C	0.7994	0.0010	1.80	11.38
DEX	Linear	0.24+0.014A-0.0075B+0.012C	0.5884	0.0172	5.42	7.08

Parameters	OND	APT	DEX
Wavelength (nm)	254	254	254
Linearity range (ng/band)	200-1200	500-1000	1000-2000
Regression coefficient(R²)	0.9997	0.9997	0.9998
Slope ±S.D^a.	2.648±0.019	4.456±0.014	2.155±0.003
%RSD^b of slope	0.746	0.326	0.167
Intercept ±S.D^a.	176.66±18.65	1395.5±54.33	449.32±60.45
Rf	0.54±0.02	0.79±0.02	0.23±0.01
LOD^c(ng/band)	23.07	29.89	92.55
LOQ^d(ng/band)	71.12	119.67	277.67

Response (Rf value)	Predicted value	Experimental value	%Residual value
APT	0.81	0.79	0.24
OND	0.54	0.56	-3.70
DEX	0.25	0.23	8.00