INTRODUCTION:

The chromatographic technique was first discovered by Russian botanist Mikhail Tsvet¹, in 1901, and has today become an indispensable tool in all the areas wherein the separation of individual chemical components is concerned. The scope of High-performance liquid chromatography (HPLC) has gained popularity in the areas of forensics, marine technology, drug discovery, toxicology food safety, and many other². Chromatography relies on a simple technique of physical separation of mixture components when passed through two phases having different polarities. One phase is a stationary (immobile)phase and the other is the mobile phase, the mobile phase is percolated over the stationary phase to bring about the separation of components in the stationary phase.

During the course, the mixture component undergoes various sorption and desorption events between mobile and immobile phases, and depending upon relevant affinity, the components of the mixture move across the stationary phase. The difference in physicochemical properties of mixture components will give different strengths of interactions between both the phases. Due to a characteristic differential transport phenomenon, the components get separated solitarily on the stationary phase³. Further advances in research and technology have catalyzed refined hybrid chromatographic methods like gas chromatography-mass spectroscopy (GC-MS), Micellar electrokinetic chromatography, and chromatography using chiral stationary phases for the separation of enantiomers^4,5. The reverse-phase high-performance liquid chromatography (RP-HPLC) contains a hydrophobic stationary phase and the polar mobile phase (but not specific to), wherein the mixture components get distributed depending upon the affinity between the two phases. Normally, the least hydrophobic component elutes first and the most hydrophobic interacts strongly with the hydrophobic stationary surface^6,7. The scope of HPLC also includes the establishment of a validated method that could explain the stability of the chemical compound under test individually or in a dosage form according to ICH guidelines⁸.

Scope of L-Arginine in medicines:

L-Arginine (L-Arg) ((2S)-2-amino-5-(diamino methylidene amino) pentanoic acid), is semi- essential amino acid. Due to its role in nitric oxide generation in the body, it is used as a drug to reduce Blood pressure. A meta-analysis of L-Arginine reports that it reduces systolic and diastolic blood pressure in hypertensive patients, but only diastolic pressure was reduced in pregnant patients⁹. It also has pro-angiogenic properties¹⁰. L-Arginine serves as a precursor for the synthesis of polyamines, proline, glutamate, creatine, agmatine, and urea^11,12, it has been researched for its role in renal failure¹³ reduction in erectile dysfunction was also reported when L-Arg alone is given for 3 months^14,15, and inhibition of gastric hyperacidity¹⁶. It is an essential component of the urea cycle. including neurotransmission, vasorelaxation, cytotoxicity, and immunity¹⁷.

As the role of L-Arg applications as nutraceuticals and as supplements is well-known. As it has been researched for a variety of ailments, alone or in combination with main drug therapy, its combinations with various other pharmaceutical agents are also expected to increase in the future. It is recently being researched as the agent to reduce blood pressure (vasodilator) in combination with cancer therapy. Polyphenolic compounds like resveratrol and its precursor piceatannol are known to have anticancer activity. The combination of L-Arg with polyphenolic compounds is emerging as a new area of interest. These polyphenolic compounds are lipophilic. A combination of L-Arg with polyphenolic compounds for the treatment of cancer can be considered to have sufficient potential to alleviate high blood pressure associated with cancer chemotherapy.

Various parametric considerations for RP-HPLC Method development:

While developing the HPLC method for analysis, quantification, or purification of chemical components, physicochemical attributes of the chemical compounds, process variables, and instrumental variables are taken into account¹⁸ (Table 1).

Table 1: Various Non-continuous attributes to consider while developing the HPLC method.

Physicochemical attributes	Electric charge, Polarity, Log K, PH, Dipole moment, H-Bond capacity, Molecular weight and size, Solubility, Volatility, Stability and Toxicity.
Process / Instrumental variables	Buffer, flow rate, column length, Temperature, pressure, mobile phase, sample preparation technique

While developing a method for a chemical moiety the first and foremost character to observe is the effect of _PH of the mobile phase on the ionization of the compound. Another technique to develop the RP-HPLC method involves the use of Quality by Design (QbD) approach which is done using the software. This exercise needs a complete literature survey to enter parameters into software to extract a computer-generated combination on which the experiment is conducted to check the reliability of the predicted combination¹⁹. Since many pharmaceutical compounds are ionizable, they tend to have different polarity, surface charge, and log D values at different _PH²⁰. To predict the behavior of ionizable compounds, one must understand the concept of _PK_a, Isoelectric point, and _PH. The ionization of the compound due to the mobile phase and _PH determines the ultimate retention and selectivity²¹^,²². This behavior can be explained by the Handerson hasselbalch equation.

The _PK_a value is a measure of acidity or the basicity of a chemical or a functional group. _PK_a is defined as the negative logarithm of the acid dissociation constant, K_a. K_a explains the dissociation of a compound to release a proton into the solution²³. So larger the K_a value greater the dissociation, and hence stronger will be the acid. As _PK_a is a negative logarithm of acid, the relation explains that the smaller the _PK_a value stronger the acid²⁴. In other words. _PK_a is the _PH at which 50% of the compound is unionized and 50% remains ionized. At any _PH below its _PK_a value, the acid remains protonated, but at _PH above _PK_a value, the acid becomes deprotonated. The value _PK_a of L-Arg is 13.8²⁵. which means it's a very weak acid and at practically all physiological _PH (up to 10) it remained essentially protonated. But like L-Arginine is triprotic amino acid, many other compounds have more than one _PK_a value²⁶ when they have more than one functional group to get protonated or deprotonated. As protonation and deprotonation are relative terms, the _PK_a value of the other two sites tends to change when one site is deprotonated. addition of a buffer in such case will conserve the change in _PH and hence we can control the protonation of a compound. So the addition of buffer solution or maintaining the _PH of the mobile phase becomes even more critical when we deal with ionizable compounds.

The isoelectric point is a value at which no net charge is present on a molecule. For L-Arg the isoelectric point is 10.6. this is particularly important when extracting amino acids in ion exchange chromatography.

The octanol-water partition coefficient (K_o/w) is also dependent upon the _PH of the medium in the case of ionizable compounds. Ideally, the P value is defined as the partition of non-ionizable compounds between octanol (organic phase) and water (aqueous phase). But when the partition of ionizable compounds comes into the picture, the distribution of ionized moiety in both phases is also considered. Hence for ionizable compounds, D-value is considered. The D value is _PH-dependent for ionizable compounds, so while selecting the mobile phase combination, considering lipophilicity will going to affect the elution²⁰.

Similarly, the ideal polarity of the mobile phase gives rise to a high eluting capacity to bring about a separation. Solute retention in the stationary phase is affected by the polarity of the mobile phase, altering the mobile phase polarity can alter the solute and stationary phase dynamics²⁷.

The selectivity also changes dramatically for polar or ionizable compounds than the non-ionizable²¹. The relationship can be studied using van’t hoffs equation²⁸. Ideally, the solvent strength and temperature do not interact with each other. But in the case of ionizable compounds, temperature, _PH, and solvent content are very peculiar²¹.

All physicochemical attributes and other noncontinuous factors like solvent system, temperature, column, buffer, etc, broadly affect the bonding between the stationary phase and mobile phase. The bonding interactions that take place during its distribution and elution are London dispersions, dipole-dipole moment, H-Bonding, and ion-dipole moment.

Strategies for method development:

The literature review suggested the use of acetonitrile, methanol, potassium dihydrogen phosphate buffer²⁹, ammonia acetate²⁹, and water, alone or in combination as mobile phase for chromatographic separation of L-Arg from the mixture. the detection is done either at 210 nm or 254nm. Using the literature review, the standard sample of L-Arg was made using 0.05M potassium dihydrogen phosphate buffer of pH 2.6 and methanol in a 50:50 ratio at 45⁰C and detected at 215nm. The peak was observed at 8.3 min at 215nm. A peak near absorbance maxima of Arginine³⁰.

To bring effective elution of L-Arginine with lipophilic polyphenolic compounds, the mobile phase was prepared using methanol and water in (70:30). The sample was prepared in 0.25mg/ml (1:9 water: methanol). Ideally, L-Arg is freely soluble in distilled water (0.5mg/ml)³¹. To solubilize L-Arginine with polyphenols water is used as cosolvent and an experiment was made to run on the above said mobile phase³². The resultant chromatogram shows different peak ranges from 2.0min to 6.1min. There could be various reasons why the different peak of the same compound appears in the chromatogram. The reasons could be the impurity of the sample itself, impurity in the injection valve, degradation of the compound, or reaction of the active compound with the solvent mixture. In the present case, the number of theoretical plates was observed to be between 100-598. This reason could affect the elution strength of the mobile phase. The chromatography column is considered to be made up of several segments or plates of height H; the magnitude of H is of the same order as the diameter of the resin particles. Within each segment, equilibrium is supposed to exist. To increase the number of theoretical plates and efficiency³³, the further trial was conducted.

Figure 1 Chromatogram of L-Arg alone in 0.05 M potassium dihydrogen phosphate buffer of pH 2.6 and methanol in 50:50 ratio.

Figure 2 Chromatogram of L-Arg in methanol and water in (70:30).

In the next trial, 0.1% OrthoPhosphoric Acid (in water): Acetonitrile (9:1) was used as the mobile phase, and a sample mixture was prepared in the solution same as the mobile phase. The peak of L-Arg was observed at 1.8 min with theoretical plates equivalent to 1108. To further increase the efficiency of the chromatogram amount of acetonitrile was increased to 30 %.

Figure 3: Chromatogram of L-Arg in 0.1% OrthoPhosphoric Acid (in water): Acetonitrile (9:1)

The final trial was conducted using 0.1% OrthoPhosphoric Acid (in water): Acetonitrile (7:3). The sample solution was also prepared using mobile phase composition. The final L-Arg peak was observed at 1.9 min and a blank peak at 2.4 mins, this was confirmed when the chromatogram was compared with the blank as a similar peak was observed at 2.4 mins.

Figure 4: chromatogram of L-Arg in 0.1% OrthoPhosphoric Acid (in water): Acetonitrile (7:3). With blank.

The peak observed thus shows good intensity. When observed for theoretical plates the count comes to be more than 2000, which is acceptable according to ICH guidelines. The various system suitability parameters were observed and mentioned in Table 2.

Table 2 System suitability parameters

Parameters	L-ARG	Acceptance Criteria
Peak Purity	0.92	Close to 1
Theoretical Plates	2144	>2000
Tailing factor	1.1	<1.5
Resolution	26.43	>20

DISCUSSION:

The elution of L-Arg through a chromatographic column was studied using four different trials. The trials were attempted strategically to bring about sufficient elution and resolution of both compounds in the mixture. Keeping all experimental parameters constant, mobile phase composition and sample preparation technique were changed and explored to bring about the suitable separation of components. In the final trial, orthophosphoric acid in a combination with Acetonitrile in the ratio of 7:3 was used as the mobile phase and the sample is also prepared using the same composition. The RT for L-Arg comes out to be at 1.9 min. The system suitability parameters were also found to be within FDA acceptance limits.

CONCLUSION:

The RP-HPLC has a vital role in drug discovery and development or wherever the quantification, purification, or detection of chemical compounds is concerned. RP-HPLC method development needs special considerations of physicochemical parameters of chemical constituents as well as non-continuous variables such as system temperature, mobile phase, column length, etc. method development for a mixture containing ionizable compound is particularly tricky since its ionization depends on the _PH of the mobile phase, its _PKa value, and other factors. The resultant net charge on the molecule defines the extent of attraction (affinity) between the stationary phase and mobile phase. Which ultimately defines the retention time on the chromatogram. The present study shares the observations and strategies applied in the RP-HPLC method developed when studied in a combination of polyphenolic compounds. Polyphenolics are primarily lipophilic and used in the treatment of cancer. The addition of L-Arg in combination therapy helps in controlling the blood pressure of the patient, occurring as a side effect of cancer therapy.

CONFLICT OF INTEREST:

The authors have no conflicts of interest to declare.

REFERENCE:

1. Gal JF. An Excursion into the History of Chromatography: Mikhail Tswett, From Asti, Italy, to Tartu, Estonia. Chromatographia. 2019; 82(2): 519-521. doi:10.1007/s10337-019-03686-0

2. Ravisankar P et al. Fundamental Chromatographic Parameters. Int J Pharm Sci Rev Res. 2019; 55(April): 46-50.

3. Chromatogrpahy. The Family of Chromatographic Techniques. In: Techniques. 1997:40-64.

4. Welch CJ. Are We Approaching a Speed Limit for the Chromatographic Separation of Enantiomers? ACS Cent Sci. 2017; 3(8): 823-829. doi:10.1021/acscentsci.7b00250

5. Pirok BWJ, Stoll DR, Schoenmakers PJ. Recent Developments in Two-Dimensional Liquid Chromatography: Fundamental Improvements for Practical Applications. Anal Chem. 2019; 91(1): 240-263. doi:10.1021/acs.analchem.8b04841

6. den Uijl MJ, Schoenmakers PJ, Pirok BWJ, van Bommel MR. Recent applications of retention modelling in liquid chromatography. J Sep Sci. 2021; 44(1): 88-114. doi:10.1002/jssc.202000905

7. Carvalho GO, da Silva CGA, Faria AM. A New Stationary Phase for Analysis of Hydrophobic Compounds by RP-LC. Chromatographia. 2016; 79(1-2): 19-27. doi:10.1007/s10337-015-2993-9

8. Garg NK, Sharma G, Singh B, Nirbhavane P, Katare OP. Quality by design (QbD)-based development and optimization of a simple, robust RP-HPLC method for the estimation of methotrexate. J Liq Chromatogr Relat Technol. 2015; 38(17): 1629-1637. doi:10.1080/10826076.2015.1087409

9. McRae MP. Therapeutic Benefits of L-Arginine: An Umbrella Review of Meta-analyses. J Chiropr Med. 2016; 15(3): 184-189. doi:10.1016/j.jcm.2016.06.002

10. Yeh CL, Pai MH, Shih YM, Shih JM, Yeh SL. Intravenous arginine administration promotes proangiogenic cells mobilization and attenuates lung injury in mice with polymicrobial sepsis. Nutrients. 2017; 9(5). doi:10.3390/nu9050507

11. Farrokhi E, Shafei MN, Khajavirad A, Hosseini M, Bideskan ARE. Role of the Nitrergic system of the cuneiform nucleus in cardiovascular responses in urethane-anesthetized male rats. Iran J Med Sci. 2017; 42(5): 473-480.

12. Ramachandran J, Daniel Peluffo R. Threshold levels of extracellular L-arginine that trigger NOS-mediated ROS/RNS production in cardiac ventricular myocytes. Am J Physiol - Cell Physiol. 2016;312(2):C144-C154. doi:10.1152/ajpcell.00150.2016

13. Cherla G, Jaimes EA. and Clinical Significance Role of L -Arginine in the Pathogenesis and Treatment of Renal Disease 1 , 2. 2018; (May):2801-2806.

14. Menafra D, de Angelis C, Garifalos F, et al. Long-term high-dose l-arginine supplementation in patients with vasculogenic erectile dysfunction: a multicentre, double-blind, randomized, placebo-controlled clinical trial. J Endocrinol Invest. 2022; 45(5): 941-961. doi:10.1007/s40618-021-01704-3

15. Barassi A, Corsi Romanelli MM, Pezzilli R, et al. Levels of L-arginine and L-citrulline in patients with erectile dysfunction of different etiology. Andrology. 2017; 5(2): 256-261. doi:10.1111/andr.12293

16. Ohta Y, Nishida K. Protective effect of L-arginine against stress-induced gastric mucosal lesions in rats and its relation to nitric oxide-mediated inhibition of neutrophil infiltration. Pharmacol Res. 2001; 43(6): 535-541. doi:10.1006/phrs.2001.0812

17. Gad MZ. Anti-aging effects of L-arginine. J Adv Res. 2010; 1(3): 169-177. doi:10.1016/j.jare.2010.05.001

18. Lunney J, Chrambach A, Rodbard D. Factors Affecting Coefficients Resolution , Plates , and Band Apparent Width , Gel Number of Theoretical Diffusion in Poiyacrylamide Electrophoresis D Y;, pz Rf when T = 0. Anal Biochem. 1971;40:158-173.

19. Karmarkar S, Garber R, Genchanok Y, George S, Yang X, Hammond R. Quality by design (QbD) based development of a stability indicating HPLC method for drug and impurities. J Chromatogr Sci. 2011; 49(6): 439-446. doi:10.1093/chrsci/49.6.439

20. Kah M, Brown CD. Log D: Lipophilicity for ionisable compounds. Chemosphere. 2008; 72(10): 1401-1408. doi:10.1016/j.chemosphere. 2008.04.074

21. Pous-Torres S, Torres-Lapasió JR, Baeza-Baeza JJ, García-Álvarez-Coque MC. Combined effect of solvent content, temperature and pH on the chromatographic behaviour of ionisable compounds. J Chromatogr A. 2007; 1163(1-2): 49-62. doi:10.1016/j.chroma.2007.06.004

22. Verma S, Mullick P, Bhatt MS, et al. Bioanalytical method development and validation for the simultaneous estimation of lamivudine and stavudine in human plasma by HPLC. Acta Pol Pharm - Drug Res. 2010; 67(4): 429-437.

23. Sharma PK, Mishra V, Verma S, Bhatia A. Simultaneous estimation by RP-HPLC method for the immunosuppressant drug combination: Mycophenolate mofetil, tacrolimus with prednisolone. Pertanika J Sci Technol. 2019; 27(1): 371-385.

24. Hanai T, Hubert J. Optimization of retention time of aromatic acids in liquid chromatography from log P and predicted pKa values. J High Resolut Chromatogr. 1984; 7(9): 524-528. doi:10.1002/jhrc.1240070906

25. Fitch CA, Platzer G, Okon M, Garcia-Moreno BE, McIntosh LP. Arginine: Its pKa value revisited. Protein Sci. 2015; 24(5): 752-761. doi:10.1002/pro.2647

26. Taylor SS, Yang J, Wu J, Haste NM, Radzio-Andzelm E, Anand G. PKA: A portrait of protein kinase dynamics. Biochim Biophys Acta - Proteins Proteomics. 2004; 1697(1-2): 259-269. doi:10.1016/j.bbapap.2003.11.029

27. Sangeetha M, Reichal R, Thirumoorthy N. Development and Validation of RP-HPLC Method: An Overview. Res Rev J Pharm Anal. 2014; 3(4): 1-7.

28. Asnin LD, Stepanova M V. Van’t Hoff analysis in chiral chromatography. J Sep Sci. 2018; 41(6): 1319-1337. doi:10.1002/jssc.201701264

29. Obreshkova DP, Tsvetkova DD, Ivanov KV. Validation of HPLC method for determination of L - Arginine in Tonotyl® solution. Int J Pharm Pharm Sci. 2012; 4(SUPPL.1): 429-432.

30. El-Bagary RI, Elkady EF, Mowaka S, Attallah MA. A validated HPLC method for simultaneous determination of perindopril arginine, amlodipine, and indapamide: Application in bulk and in different pharmaceutical dosage forms. J AOAC Int. 2017; 100(4): 992-999. doi:10.5740/jaoacint.16-0279

31. Bowden NA, Sanders JPM, Bruins ME. Solubility of the Proteinogenic α-Amino Acids in Water, Ethanol, and Ethanol-Water Mixtures. J Chem Eng Data. 2018; 63(3): 488-497. doi:10.1021/acs.jced.7b00486

32. Ishida Y, Fujita T, Asai K. New detection and separation method for amino acids by high-performance liquid chromatography. J Chromatogr A. 1981; 204(C): 143-148. doi:10.1016/S0021-9673(00)81650-7

33. Ceulemans J. The Number of Theoretical Plates at Infinite Capacity: A Better Measure of the Efficiency of Gas Chromatographic Columns. J Chromatogr Sci. 1984; 22(7): 296-299. doi:10.1093/chromsci/22.7.296

Received on 10.08.2022 Modified on 28.01.2023

Research J. Pharm. and Tech 2024; 17(1):115-119.

DOI: 10.52711/0974-360X.2024.00018