Int J Pharm Pharm Sci, Vol 12, Issue 9, 41-48Original Article

RP-HPLC METHOD DEVELOPMENT AND VALIDATION FOR THE COMBINATION OF IMIQUIMOD AND SALICYLIC ACID

ANKITA SHARMA1*, INDER KUMAR2, KARAN RANA3

1*Department of Pharmaceutical Chemistry, Abhilashi College of Pharmacy, Nerchwok, Mandi, HP, India, 2Department of Pharmaceutics, School of Pharmacy, Abhilashi University, Mandi, HP, India, 3Quality Control Executive, Complex Pharma, Karnal, Haryana, India
Email: [email protected]

Received: 19 May 2020, Revised and Accepted: 14 Jul 2020


ABSTRACT

Objective: The present study was undertaken to develop and validate an RP-HPLC method for the combination of imiquimod and salicylic acid

Methods: The method was carried out on Nucleodur C18 (250 mm × 4.6 mm I.D., 5 𝜇m) using low-pressure gradient elution mode. The mobile phase was used as 30M potassium dihydrogen phosphate and acetonitrile (45:55) pH 6.5 adjusted using ortho-phosphoric acid. The concentration of solvents was 1-20 µg/ml and the volume of injection was 20 mcl with the flow rate of 1.0 ml/min. The absorption maxima of salicylic acid and imiquimod were found 234 nm and 226 nm, respectively.

Results: The method was validated and showed the linearity greater than 0.99% and with precision (RSD%<1). The limit of detection (LOD) and limit of quantification (LOQ) of salicylic acid was found to be 0.09756 µg/ml and 0.2956 µg/ml, respectively, and imiquimod was found to be 0.044031 µg/ml and 0.13334 µg/ml, respectively.

Conclusion: The method developed in the present study was found to be sensitive, specific, and can be applied for the simultaneous estimation of imiquimod and salicylic acid.

Keywords: Imiquimod, Salicylic acid, Reverse-phase high-performance liquid chromatography (RP-HPLC), Validation, International council for harmonization (ICH)


INTRODUCTION

Imiquimod is chemically 1-(2-methyl propyl)-1H-imidazo [4, 5-c] quinolin-4-amine, which is a novel synthetic compound that is a member of the imidazoquinolone family of drugs [1]. The molecular formula of imiquimod is C14H16N4, which is a white crystalline powder and the molecular weight is 240.30g/mol. The solubility of imiquimod is freely soluble in oleic acid and lactic acid with melting point 292-294 °C and pKa value 2.7. Imiquimod is an immune response modifiers use in the treatment for external genital warts by the mechanism response modifier that acts as a toll-like receptor 7 agonists. It does not kill the viruses that cause warts directly, however, also used to treat skin problems of face and scalp that is called actinic keratoses and also for various skin cancers called superficial basal cell carcinoma. In a present-day commercially available as Aldara® 5% imiquimod cream, approved by U. S. Food and Drug Administration (“FDA”) in 1997 [2-4].

Fig. 1: Chemical structure of imiquimod

Salicylic acid (from latin salix, willow tree, from the bark of which the substance used to be obtained) is a monohydroxybenzoic acid, a type of phenolic acid and a beta hydroxy acid. Salicylic acid has the formula C6H4(OH) COOH, where the OH group is ortho to the carboxyl group and is poorly soluble in water (2 g/l at 20 °C). Aspirin (acetylsalicylic acid or ASA) can be prepared by the esterification of the phenolic hydroxyl group of salicylic acid with the acetyl group from acetic anhydride or acetyl chloride. The compound is white crystalline powder and the molecular weight is 138.12 g/mol. The solubility of salicylic acid is good in ether, CCl4, benzene, propanol, acetone, ethanol, oil of turpentine, toluene [5, 6]. It shows its anti-inflammatory action by suppressing the activity of cyclooxygenase (COX), an enzyme that is responsible for the production of pro-inflammatory mediators such as the prostaglandins [7]. Salicylic acid also has its anti-acne action [8]. Chemical structure of salicylic acid is given below:

Fig. 2: Chemical structure of salicylic acid

Simultaneously steps were taken for these two drugs successfully. Therefore it was thought to develop an accurate and rapid RP-HPLC method for simultaneous estimation of the combination of imiquimod and salicylic acid. This method was validated as per the current International Conference on Harmonization (ICH) guideline [9-11].

MATERIALS AND METHODS

Salicylic acid was procured as a gift sample from SGPTC Pvt. Ltd and imiquimod drug was obtained as a gift sample from Glenmark Pharma, Mumbai India. All other chemicals like o’phosphoric acid, potassium dihydrogen phosphate, and disodium hydrogen orthophosphate were used of AR grade and solvents like methanol, acetonitrile, and water were used of HPLC grade

Identification of standard drugs

The identification of standard drugs was carried out by melting point study, infrared spectroscopic study, and solubility study.

Rp-hplc method

Selection of detection wavelength

A known concentration of 10 μg/ml of salicylic acid and imiquimod were prepared in methanol and scanned in the UV region 200-400 nm.

Selection of mobile phase and optimization of chromatographic condition

Initially, the mobile phase was tried with methanol: water, acetonitrile: water, acetonitrile: water: methanol and then finally with phosphate buffer: acetonitrile: at various pH conditions. The following optimized parameters were used as a final method for the simultaneous estimation of salicylic acid and imiquimod.

Chromatographic condition

In the optimized parameters, the stationary phase was Nucleodur C18 (250 mm × 4.6 mm I.D., 5 𝜇m) using low-pressure gradient elution mode. 30M potassium dihydrogen phosphate and acetonitrile (45:55) pH 6.5 were used as mobile phase and pH were adjusted using orthophosphoric acid. The concentration of solvents was 1-20 µg/ml, and the volume of injection was 20 µl with the flow rate of 1.0 ml/min. The absorption maxima of salicylic acid and imiquimod were set in 234 nm and 226 nm, respectively.

Standard stock solution preparation (100 μg/ml)

100 μg/ml solution was prepared using 10 mg of salicylic acid 1 mg of imiquimod and about 1 ml of diluent was added and sonicate to dissolve to make the standard solution.

Preparation of calibration curve

Different concentration was prepared 0.1 μg/ml to 1 μg/ml for imiquimod and 1 µg/ml to 10 µg/ml for salicylic acid from the standard stock solution, respectively.

Validation of RP-HPLC method

Linearity

Different concentrations of both the drugs were prepared for linearity i.e. salicylic acid 1-10 μg/ml and imiquimod 0.1-1 μg/ml [12, 13].

Accuracy

Recovery studies were carried out by the addition of standard drug solution at the level of 80%, 100%, and 120% to the pre-analyzed sample. Results of the recovery study were found to be within the acceptance criteria 100±10 %. In this method, the known concentration of standard drug was added to the assay sample [14, 15].

Precision

Different concentrations of both the drugs were used for intra-day and inter-day variation for six times in the same day. The concentration was 5 μg/ml of salicylic acid and 0.5 μg/ml imiquimod, and then % RSD was calculated [16, 17].

Limit of detection and limit of quantification

The limit of detection can be calculated using the following equation as per ICH guidelines [18].

Where N is the standard deviation of the intercepts of the drug, and S is the slope of the corresponding calibration curve.

The limit of quantification can be calculated using the following equation as per ICH guidelines.

Where N is the standard deviation of the intercepts of the drug, and S is the slope of the corresponding calibration curve [19].

Robustness

Same samples of salicylic acid and imiquimod concentration 5μg/ml and 0.5μg/ml respectively by considered variation in the method parameters i.e. change in wavelength or change in flow rate, change in pH of the mobile phase. The change in the responses of salicylic acid and imiquimod were noted in terms of %RSD [20].

Specificity

The specificity of the HPLC method was demonstrated by the separation of the analytes from other potential components such as impurities, degradants, or excipients. A volume of 20μl of individual ingredients and excipients solution was injected and the chromatogram was recorded [21].

Ruggedness

The ruggedness was studied by analyzing the same samples of salicylic acid and imiquimod concentration 5μg/ml and 0.5μg/ml, respectively by changing the analyst. The change in the responses of salicylic acid and imiquimod were noted in terms of %RSD [22].

RESULTS AND DISCUSSION

Identification of imiquimod and salicylic acid

Salicylic acid and imiquimod were observed for organoleptic properties like physical appearance, odor, and melting point. The drugs were identified with the help of UV and FTIR, and exhibited absorption maxima was 234 nm for salicylic acid and 226 nm for imiquimod when methanol was used as a solvent as mentioned in the literature (fig. 3).

Fig. 3: UV spectrum of salicylic acid (A) and imiquimod (B) in methanol

(a)

(b)

Fig. 5: FTIR spectra of (a) salicylic acid (b) imiquimod

Method validation

Linearity

The linearity of both the drugs was found within acceptance criteria. The correlation coefficient (r2) obtained was calculated and it was found to be greater than 0.99 for salicylic acid and imiquimod is given in the below table, which is well within the acceptance criteria. Good linearity with the coefficient of correlation 0.99 indicated that the proposed method was linear within the range of 1 to 10 µg/ml, 0.1 to 1 µg/ml, respectively for salicylic acid and imiquimod.

Table 1: Linearity of salicylic acid

Conc.(µg/ml) Area1 Area2 Area3 Mean SD %RSD
1 55346 55051 55866 55421 412.6439 0.744562
2 94156 94907 94550 94537.67 375.6519 0.397357
3 129106 125734 124812 126550.7 2260.491 1.786234
4 162548 165085 167974 165202.3 2714.902 1.64338
5 187989 189886 194048 190641 3099.256 1.625703
6 230434 236106 231690 232743.3 2979.099 1.279993
7 267238 268739 264993 266990 1885.274 0.706121
8 304641 308556 307314 306837 2000.613 0.652012
9 342474 341383 348660 344172.3 3924.53 1.14028
10 383247 388043 386828 386039.3 2493.371 0.645885

*Conc. = concentration, mean±SD, n=3, SD = standard deviation, RSD = relative standard deviation

Table 2: Linearity of imiquimod

Conc.(µg/ml) Area1 Area 2 Area 3 Mean SD %RSD
0.1 100795 100900 100854 100849.7 52.63396 0.052191
0.2 165045 165650 164071 164922 796.6536 0.483049
0.3 225478 220799 227558 224611.7 3461.78 1.541229
0.4 268053 266795 267947 267598.3 697.723 0.260735
0.5 330441 336627 332784 333284 3123.163 0.937088
0.6 395254 394840 397691 395928.3 1540.485 0.389082
0.7 468121 469870 463485 467158.7 3299.488 0.706288
0.8 522375 524480 525548 524134.3 1614.496 0.308031
0.9 595412 595953 595245 595536.7 370.0977 0.062145
1 655536 652236 651048 652940 2325.349 0.356135

*Conc. = concentration, mean±SD, n=3, SD = standard deviation, RSD = relative standard deviation

Fig. 6: Average linearity of salicylic acid (left) and imiquimod (right)

Fig. 7: Chromatogram of salicylic acid and imiquimod (4 μg/ml and 0.4 μg/ml)

Table 3: Accuracy results of salicylic acid and imiquimod

Accuracy results of salicylic acid
Conc. level Area Mean Amount added Amount recovered % Recovery SD %RSD
80% 167045 165052.66 4 µg/ml 4.12 103.055 2172.9 1.3165
163972 4 µg/ml 4.03 100.932
164141 4 µg/ml 4.04 101.049
100% 183748 184728.33 5 µg/ml 4.58 91.674 882.13 0.4775
185458 5 µg/ml 4.63 92.619
184979 5 µg/ml 4.61 92.354
120% 215834 217098.66 6 µg/ml 5.47 91.170 1362.2 0.6275
216921 6 µg/ml 5.50 91.671
218541 6 µg/ml 5.54 92.417
Accuracy results of imiquimod
Conc. level Area Mean Amount added Amount recovered % Recovery SD %RSD
80% 261795 261930.33 0.4 µg/ml 0.369 92.39 685.89 0.2619
260825 0.4 µg/ml 0.367 91.99
263171 0.4 µg/ml 0.371 92.95
100% 319439 321252.33 0.5 µg/ml 0.463 92.67 1936.4 0.6028
321026 0.5 µg/ml 0.465 93.18
323292 0.5 µg/ml 0.469 93.92
120% 375617 375861 0.6 µg/ml 0.555 92.45 1236.2 0.3289
377201 0.6 µg/ml 0.557 92.88
374765 0.6 µg/ml 0.553 92.22

*Conc. = concentration, mean±SD, n=3, SD = standard deviation, RSD = relative standard deviation

Fig. 8: Chromatogram of salicylic acid and imiquimod (80%)

Accuracy

The results indicate that the recoveries are well within the acceptance range of 80–120% (table 3). The chromatogram of salicylic acid and imiquimod drugs at level 80% was shown in fig. 8.

Precision

The %RSD for the area of six replicate injections was found to be within the specified limits i.e. for salicylic acid, it was 0.742393% and imiquimod it was observed 0.995204%. Whereas the % RSD for intra and inter-day precision of salicylic acid and imiquimod were observed below 2% [18-20]. The low values of % RSD indicate that the method is precise. % RSD for interday precision and intraday precision was given below that was found to be within the specified limits.

Limit of detection and limit of quantitation

The LOD was found to be 0.09756 µg/ml and 0.044031 µg/ml for salicylic acid and imiquimod respectively and LOQ was found to be 0.2956 µg/ml and 0.13334 µg/ml salicylic acid and imiquimod respectively which showed that sensitivity of the method was high.

Robustness

The Percentage of RSD should not be more than 2. The %RSD obtained for change of flow rate, change in wavelength, and pH variation in the mobile phase was found to be below 2, which was within the acceptance criteria. Hence the method was robust.

Ruggedness

The results were found within a specified limit, % RSD was less than 2. Data are tabulated in table 9 and fig. 10.

Specificity

Specificity of salicylic acid and imiquimod are shown in fig. 11 and table 10. All the data were within limits. %RSD of salicylic acid in specificity was observed 0.186425% and for imiquimod, it was 0.384456%.

Table 4: Repeatability of salicylic acid and imiquimod

S. No. Salicylic acid Imiquimod
Conc.(µg/ml) Area Conc.(µg/ml) Area
1 5 180729 0.5 312844
2 5 180281 0.5 318685
3 5 181842 0.5 311867
4 5 182989 0.5 310441
5 5 183886 0.5 316627
6 5 182048 0.5 312784
Mean 181962.5 Mean 313874.7
Standard Deviation 1350.877 Standard Deviation 3123.692
%RSD 0.742393 %RSD 0.995204

*Mean for six independent analysis, mean±SD, n=6, Conc. = concentration, RSD = relative standard deviation

Table 5: Intraday and intraday precision of salicylic acid and imiquimod

Intraday precision
S. No. Salicylic acid Imiquimod
Conc.(µg/ml) Area Conc.(µg/ml)
1 5 194588 0.5
2 5 194165 0.5
3 5 195142 0.5
4 5 194057 0.5
5 5 195544 0.5
6 5 194999 0.5
Mean 194749.2 Mean 313874.7
Standard Deviation 582.2053 Standard Deviation 3123.692
%RSD 0.298951 %RSD 0.995204
Interday precision
S. No. Salicylic acid Imiquimod
Conc.(µg/ml) Area
1 5 192990 1
2 5 193563 2
3 5 193229 3
4 5 193825 4
5 5 193199 5
6 5 193807 6
Mean 193435.5 Mean 369890.8
Standard Deviation 347.2819 Standard Deviation 2046.481
%RSD 0.179534 %RSD 0.553266

*Mean for six independent analysis, mean±SD, n=6, RSD = relative standard deviation

Table 6: Limit of detection and quantitation

API parameter Linearity 1 Linearity 2 Linearity 3 Intercept SD Slope mean LOD LOQ
Salicylic acid Intercept 18267 19774 17719 1064.14112 35992.3333 0.09756 µg/ml 0.2956 µg/ml
Slope 35900 35722 36355
Imiquimod Intercept 34160 34149 35575 820.144499 61466.3333 0.044031 µg/ml 0.13334 µg/ml
Slope 61543 61575 61281

*mean±SD, n=3, LOD = limit of detection, LOQ = limit of quantitation

Fig. 9: Chromatogram of repeatability of salicylic acid and imiquimod

Table 7: Robustness of salicylic acid and imiquimod at different flow rates and column temperature

Change in flow rates
Conc.(µg/ml) 0.690 ml/min 0.700 ml/min 0.710 ml/min
Area Area Area
Salicylic acid Imiquimod Salicylic acid Imiquimod Salicylic acid Imiquimod
5, 0.5 185355 336750 185716 331298 179209 322635
5, 0.5 186835 331907 185978 327514 180008 319692
5, 0.5 184881 331433 185234 332858 181071 323943
Mean 185690.333 333363.33 185642.7 330556.7 180096 322090
SD 1019.24743 2942.4993 377.3822 2748.048 934.114 2177.273
%RSD 0.54889634 0.8826704 0.203284 0.831339 0.518676 0.675983
Change in column temperature
Conc.(µg/ml) 35 °C 35 °C 35 °C
Area Area Area
Salicylic acid Imiquimod Salicylic acid Imiquimod Salicylic acid Imiquimod
5, 0.5 183450 326827 189534 366900 190840 366438
5, 0.5 182884 325182 194165 366835 193563 366114
5, 0.5 187570 326333 193229 369038 195142 369995
Mean 184634.667 326114 192309.3 367591 193181.66 367515.7
SD 2557.77742 844.0835 2448.649 1253.56 2176.2036 2153.268
%RSD 1.38531808 0.258831 1.273286 0.34102 1.1265063 0.585898

*Conc. = concentration, mean±SD, n=3, SD = standard deviation, RSD = relative standard deviation

Table 8: Robustness of salicylic acid and imiquimod at different mobile phase ratio and different wavelength

Conc.(µg/ml) Buffer: ACN 49.9:50.1 Buffer: ACN 50.1:49.9 Buffer: ACN 50:50
Area Area Area
Salicylic acid Imiquimod Salicylic acid Imiquimod Salicylic acid Imiquimod
5, 0.5 192990 371471 193807 370423 189534 366900
5, 0.5 194999 369934 194057 370772 194165 366835
5, 0.5 195544 368306 189264 368613 193229 369038
Mean 194511 369903.7 192376 369936 192309.3 367591
SD 1345.11598 1582.718 2697.968 1158.964 2448.648 1253.56
%RSD 0.69153723 0.427873 1.402445 0.313288 1.273286 0.34102
Change in wavelength
Conc.(µg/ml) 222 nm 227 nm 232 nm
Area Area Area
Salicylic acid Imiquimod Salicylic acid Imiquimod Salicylic acid Imiquimod
5, 0.5 167812 287540 185799 320120 147045 394888
5, 0.5 168691 286264 184977 319351 147211 388641
5, 0.5 167785 287674 183279 321442 146878 392983
Mean 168096 287159.3 184685 320304.3 147044.66 391764.5
SD 515.46192 778.2707 1285.126 1057.617 166.50025 4417.296
%RSD 0.3066473 0.271024 0.695847 0.330191 0.11323107 1.127539

*Conc. = concentration, mean±SD, n=3, ACN = acetonitrile, SD = standard deviation, RSD = relative standard deviation

Table 9: Ruggedness study by analyst 1 and analyst 2

Conc.(µg/ml) Analyst 1 Analyst 2
Area Area
Salicylic acid Imiquimod Salicylic acid Imiquimod
5, 0.5 193825 371153 203497 386815
5, 0.5 194588 371303 198780 384112
5, 0.5 193199 371146 196176 380535
Mean 193870.6667 371200.6667 199484.3333 383820.6667
SD 695.6251385 88.69235223 3710.973502 3150.120051
%RSD 0.358808865 0.023893371 1.860283181 0.820727054

*Conc. = concentration, mean±SD, n=3, SD = standard deviation, %RSD = relative standard deviation

(A) (B)

Fig. 10: Chromatogram of salicylic acid and imiquimod by analyst 1 (A) and analyst 2 (B)

Table 10: Specificity data of salicylic acid and imiquimod

Conc.(µg/ml) Specificity
Area
Salicylic acid Imiquimod
5, 0.5 189068 336047
5, 0.5 188871 335741
5, 0.5 189556 333678
Mean 189165 335155.3
SD 352.6514 1288.524
%RSD 0.186425 0.384456

*Conc. = concentration, mean±SD, n=3, SD = standard deviation, %RSD = relative standard deviation

Fig. 11: Chromatogram of salicylic acid and imiquimod (specificity)

CONCLUSION

The RP-HPLC method was carried out on Nucleodur C18 (250 mm × 4.6 mm I.D., 5 𝜇m) using low-pressure gradient elution mode. The limit of detection (LOD) and limit of quantification (LOQ) of salicylic acid was found to be 0.09756 µg/ml and 0.2956 µg/ml, respectively, and imiquimod was found to be 0.044031 µg/ml and 0.13334 µg/ml, respectively. The developed and validate method for imiquimod and salicylic acid was found to be sensitive, specific, and accurate and can be effectively applied for the simultaneous estimation of imiquimod and salicylic acid.

ACKNOWLEDGMENT

The Author is highly grateful to Dr. R. K Abhilashi, Chairman Abhilashi Group of Institutions, Mandi, India, for providing necessary facilities to carry out the research.

FUNDING

Nil

AUTHORS CONTRIBUTIONS

All the author has contributed equally.

CONFLICT OF INTERESTS

Declared none

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