INTRODUCTION:

L-methylfolate is chemically referred to as (2S)-2-[[4-[(2-Amino-5-methyl-4-oxo-1, 6,7,8-tetrahydropteridin-6-yl) methylamino [benzoyl] amino] pentane dioic acid L-methyl folate is used to form methionine and tetrahydrofolate (THF) for the methylation of homocysteine. THF is the immediate acceptor for the synthesis of thymidine-DNA, purines (RNA and DNA) and methionine one-carbon units. The non-methylated form, folic acid (vitamin B9), is a synthetic folate form and must undergo enzymatic reduction to become biologically active by methylenetetrahydrofolate reductase (MTHFR)^1-3.

The only folate metabolite that can cross the blood-brain barrier is L-methylfolate, and it is this type that can directly affect many significant CNS reactions, most notably the synthesis of three main neurotransmitters: serotonin, epinephrine and dopamine. Structure of the L-MF were shown in figure 1.⁴

Fig. 1: Structure of L-Methyl Folate

Tofisopam is a 2,3-benzodiazepine that is structurally similar to, and shares some of the same actions with, 1,4 benzodiazepines, such as diazepam. However, the selective, anticonvulsant, and muscle relaxant properties of traditional benzodiazepine are stated to be largely missing.⁵Tofisopam is sold in certain European countries for the treatment of anxiety. The compound does not bind to the benzodiazepine binding site of the γ-aminobutyric acid receptor, as opposed to classical 1,4-benzodiazepines, and its psychopharmacological profile varies from that of such compounds. There are records of antipsychotic effects in addition to anxiolytic properties. Without sedative-hypnotic or muscle relaxant effects, it is an anxiolytic. Tofisopam, as other benzodiazepines do, does not affect psychomotor and intellectual performance. It has a moderate cognitive stimulatory function, on the other hand. Tofisopam was prescribed orally for short-term therapy. It can be inferred that dual or triple inhibition of PDE isoenzymes with additive or synergistic effects can be an interesting approach to pharmacological action, as shown by the long-term use of tofisopam in patients.⁶The R-(+) isomer dextrofisopam is being investigated in the treatment of irritable bowel syndrome.⁷

Fig. 2: Structure of Tofisopam

With the implementation of modern pumping techniques, more powerful columns and a wide range of detectors, HPLC has been rapidly developed. Knowledge of its molecular weight, polarity, ionic character, pKa values, absorption wavelength, compound purity and solubility must be understood in order to create a new HPLC process for any drug.^8,9for the estimation of L-methylfolate and Tofisopam in pharmaceutical dosage form, The objective of this research was to develop and validate a process of HPLC that was sensitive, accurate, precise and specific.

MATERIALS AND METHODS:

Instrumentation:

Analysis was carried out on the Waters alliance-2695 chromatographic system, equipped with a quaternary pump and PDA detector-2996.¹⁰ for data collection and processing, chromatographic software Empower-2.0 was used.

Reagents and chemicals:

Merk (India) Ltd. purchased acetonitrile (HPLC grade), orthophophoric acid (HPLC grade), and water (HPLC grade). Worli, in Bombay, India. Supriya Life Sciences, Goregaon (E), Mumbai, India (99.7-99.9 percent purity) procured all active pharmaceutical intergradient’s (APIs) of L-Methyl Folate and Tofisopam as reference standards.

Chromatographic Terms:

Using Isocratic elution and Acetonitrile, chromatographic analysis was carried out: 0.1 percent PH modified to 2.5 with OPA (50:50 by volume) as a mobile phase and filtered through 0.45μ membrane filter paper. The mobile phase flow rate was monitored at 1 ml/min¹¹and eluents were detected at 234nm. 3000 psi operating pressure was maintained at room temperature by injecting 10μl volume with 5 minutes of runtime.

Wavelength Selection:

The absorption spectra of the solution of the two drugs in acetonitrile were scanned in the region 200nm-400nm¹²against acetonitrile as blank using the Photodiode spectrophotometer, and the spectra are shown in the figure. From the figure, the L-Methyl Folate and Tofisopam spectra show the same λmax viz. of 232nm. 227nm was chosen as the detection wavelength for HPLC Chromatographic method by considering the chromatographic parameter, sensitivity and process selectivity for three medications.

Fig. 3: PDA Spectrum for L-Methyl Folate and Tofisopam

Sample Solution Preparation:

Weigh 20 tablets and take the corresponding weight of one tablet. Crush the 10 tablets into powder form, take 200mg of sample into a 100ml volumetric flask¹³ and apply 70ml of mobile phase to the target with mobile phase for 30min after that. Further dilute 5ml of the above solution to make up to 50ml volumetric flask with mobile phase label. Filter into the filtre of a 0.45μ nylon syringe.

Validating:

The aim of the optimized chromatographic separation was to achieve a resolution above 1.5 between all components, the tailing factor is less than 2.0, and the plate count will be more than 2000 w.r. to the stationary, mobile phase compositions, and flow rate, volume of sample, wavelength and temperature detection.

RESULTS AND DISCUSSIONS:

Method development and optimization:

For both the L-Methyl Folate and Tofisopam drugs picked,Appropriateness, linearity, accuracy, precision, robustness, and precision In this system frame function, forced degradation trials are validated.

Fig. 4: Standard Chromatogram of L-Methyl Folate

Fig. 5: Standard Chromatogram of Tofisopam

System suitability:

Standard solution is prepared in the concentration of L-Methyl Folate 100μg/ml and Tofisopam 40μg/ml was found to be 4.335 and 7.683 min respectively. Resolution of the Tofisopam was 8.36 from the L-Methyl Folate. The number of theoretical plate counts for L-Methyl Folate and Tofisopam were same 5876. All the parameters found to be within the limit.μμ

Fig. 6: Typical Chromatogram for System Suitability of L-Methyl Folate

Table 1: Results for suitability of the L-Methyl Folate process

Injection	Retention time (min)	Peak area	Theoretical plates (TP)	Tailing factor (TF)
1	5.696	2879172	5880	0.99
2	5.754	2878655	5865	0.98
3	5.654	2878498	5978	0.99
4	5.652	2870566	5902	0.99
5	5.6933	2876583	5864	0.99
6	5.754	2873838	5905	0.99
Mean	5.686	2871220	5876	0.98
SD	0.1248	4301.16	--	--
%RSD	0.11	0.2816	--	--

Table 2: Results for suitability of the Tofisopam process

Injection	Retention time (min)	Peak area	Theoretical plates (TP)	Tailing factor (TF)
1	3.396	2879172	5880	0.99
2	3.354	2878655	5865	0.98
3	3.354	2878498	5978	0.99
4	3.352	2870566	5902	0.99
5	3.3933	2876583	5864	0.99
6	3.324	2873838	5905	0.99
Mean	3.386	2871220	5876	0.98
SD	0.1248	4301.16	--	--
%RSD	0.11	0.2816	--	--

Fig. 7: Typical Chromatogram for System Suitability of Tofisopam

Fig. 8: Linearity of detector response graphs for L-Methyl Folate

Table 3: Detector Response Linearity for L-Methyl Folate

S. No.	Conc.(µg/ml) of L-Methyl Folate	Area	Acceptance criteria
S. No.	Conc.(µg/ml) of L-Methyl Folate	L-Methyl Folate	Acceptance criteria
1	10.0 µg/ml	284986	Squared co relation coefficient should be not less than0.999.
2	25 µg/ml	718827
3	50 µg/ml	1430498
4	100 µg/ml	2872400
5	125.0 µg/ml	3591227
6	150 µg/ml	4302898

Table 4: Detector Response Linearity for Tofisopam

S. No.	Conc.(µg/ml) of Tofisopam	Area	Acceptance criteria
S. No.	Conc.(µg/ml) of Tofisopam	Tofisopam	Acceptance criteria
1	5.0 µg/ml	284986	Squared co relation coefficient should be not less than0.999.
2	12.5 µg/ml	718827
3	25 µg/ml	1430498
4	50 µg/ml	2872400
5	62.5 µg/ml	3591227
6	75 µg/ml	4302898

Linearity:

The linearity of the analytical method is its capacity (within a given range) to achieve test results that are directly proportional to the sample's analytical concentration (amount). In the range of 10 percent-150 percent test concentration of 450mg tablets, a series of L-Methyl Folate and Tofisopam standard solutions were prepared and injected into the HPLC system as per the test process.

Fig. 9: Linearity of detector response graphs for Tofisopam

Precision:

The precision of the analytical method represents the closeness of agreement (degree of dispersion) between the series of measurements obtained under the specified conditions from multiple samples of the same homogeneous sample.¹⁴

Table 5: Method precision data for L-Methyl Folate

L-Methyl Folate
S. No.	Rt	Area
1	5.505	2918923
2	5.495	2917262
3	5.498	2926996
4	5.499	2916363
5	5.497	299401
6	5.507	2919405
Avg	5. 508	2964725
St dev	0.012	182112
%RSD	0.05	1.4210

Table 6: Method precision data for Tofisopam

Tofisopam
S.No.	Rt	Area
1	3.305	2918923
2	3.325	2917262
3	3.318	2926996
4	3.329	2916363
5	3.327	299401
6	3.307	2919405
Avg	3. 308	2964725
St dev	0.012	182112
%RSD	0.05	1.4210

Fig. 10: Typical chromatogram for precision for L-Methyl Folate

Fig. 11: Typical chromatogram for precision for Tofisopam

Accuracy:

An analytical procedure’s accuracy reflects the closeness of agreement between the value that is accepted either as a conventional true value or as an accepted reference value and the found value. Often this is called trueness. A triplicate sample solution series was prepared (3 preparations for L-Methyl Folate and Tofisopam levels 50 percent to 150 percent by spiking the L-Methyl Folate and Tofisopam API on placebo in the range of approximately 50 percent to 150 percent of the 40 mg tablet test concentrations, injected into the HPLC system and analyzed according to the test method). Three calculations were carried out at each stage.¹⁵

Individual percent recovery, mean percent recovery, percent RSD and square correlation coefficient were determined for the test method's linearity and the results were found to be within the acceptable limits.

Table 7: Accuracy data of L-Methyl Folate

Recovery level	Accuracy L-Methyl Folate						%RSD
	Amount taken(µg/ml)	Area	%Assay	Average area	Amount recovered (µg /ml)	% Recovery
50%	50.0	1480583	100.5	1485510	100.58	100.5	0.18
	50.0	1488385	100.1
	50.0	1484635	100.4
100%	100.0	2870991	100.8	2873832	100.64	100.6	0.62
	100.0	2923255	100.6
	100.0	2910832	100.3
150%	150.0	4300434	100.5	4302945	100.52	100.7	0.26
	150.0	4301334	100.8
	150.0	4304702	100.1

Table 8: Accuracy data of Tofisopam

Recovery level	Accuracy Tofisopam						%RSD
	Amount taken(µg/ml)	Area	%Assay	Average area	Amount recovered ( µg /ml)	%Recovery
50%	25.0	1480583	100.5	1485510	100.58	100.5	0.18
	25.0	1488385	100.1
	25.0	1484635	100.4
100%	50.0	2870991	100.8	2873832	100.64	100.6	0.62
	50.0	2923255	100.6
	50.0	2910832	100.3
150%	75.0	4300434	100.5	4302945	100.52	100.7	0.26
	75.0	4301334	100.8
	75.0	4304702	100.1

Fig. 12: Typical chromatogram for accuracy for L-Methyl Folate

Fig. 13: Typical chromatogram for accuracy for Tofisopam

Robustness:

An analytical procedure's robustness is a measure of its ability to remain unaffected by minor, but deliberate, variations in the parameters of the system¹⁶ and provides an indicator of its reliability during normal use.

Variation in flow rate:

A research was conducted to determine the impact of flow rate variance. Standard and control standard solutions with flow rates of 1.0ml/min were prepared according to the test method and injected into the HPLC system. The parameters of system suitability were assessed and found to be within the specified limits according to the test method and the main peak RT was monitored.

Variation in the organic phase:

A study was conducted to determine the effect of organic phase variation. Standard and control standard solutions were prepared according to the test method and injected into the HPLC device with a ratio of 50:50 percent v / v and 232nm of mobile phase water and ACN. Device suitability parameters were found to be within the defined limits and 50:50 percent of the RT of the main peak was monitored.

Table 9: Flow rate and Organic phase variations of L-Methyl Folate

Parameters	L-Methyl Folate
Flow rate	Retention Time	Tailing factor	% RSD
0.8ml/min	7.384	1.09	1.06
1.0ml/min	5.772	0.89	1.12
1.2ml/min	4.754	1.26	1.16
Organic phase
85:15	6.260	0.94	0.89
90:10	5.776	1.03	1.18
95:5	5.351	1.11	1.41

Fig. 14: Typical chromatogram of L-Methyl Folate robustness

Table 10: Flow rate and Organic phase variations of Tofisopam

Parameters	Tofisopam
Flow rate	Retention Time	Tailing factor	% RSD
0.8ml/min	3.835	1.09	1.06
1.0ml/min	3.328	0.89	1.12
1.2ml/min	2.925	1.26	1.16
Organic phase
45:55	4.044	0.94	0.89
50:50	3.329	1.03	1.18
55:45	2.884	1.11	1.41

Forced degradation studies:

Drug product, placebo formulation containing one drug substance and blank, forced degradation studies were carried out by applying the following types of stress to degradation of about 1% to 20% Peak purity degradation table for samples of forced degradation.¹⁷

Fig. 15: Typical chromatogram of Tofisopam robustness

Acid degradation:

To this add 2ml 1N HCl and kept on the bench top for 10min, 350mg of sample transferred into 50ml volumetric flask then neutralized it with 2ml of 1N NaOH, then the remaining procedure is the same as the test preparation.

Base degradation:

To this add 2ml 1N NaOH and kept on the bench top for 10min, 350mg of sample transferred into 50ml volumetric flask then neutralized with 2ml of 2N HCl, then the remaining procedure is the same as the preparation of the test.

Degradation with peroxide:

To this, 350mg of sample into a 50ml volumetric flask add 30 percent H₂O₂ and keep on the bench top for 10 minutes, and then the remaining procedure is the same as the preparation for the test.

Degradation-reduction:

To this add 1ml 10 percent Sodium bi sulphate solution, 350mg of sample into 50ml volumetric flask and kept on the bench top for 10 minutes, then the remaining procedure is the same as the preparation for the test.

Degradation thermal:

API was subjected to heat 700c for 20 minutes for the respective weights of the sample, then the samples were analyzed as test preparation.

Degradation of Hydralysis:

Add 1 ml of water to this 350mg of sample into a 50ml volumetric flask and keep on the bench top for 10 minutes, and then the remaining procedure is the same as the preparation for the test.

Degradation by Photolytic:

The sample was exposed to 90 mins of sunlight and analyzed with the exposed sample.

Table 11: Degradation Studies Data of L-Methyl Folate

S. No	Degradation Parameters	Time	Peak Area	% Recovery	% Degradation
1	Acid	10 min	2306402	78.20	21.25
2	Alkali	10 min	2304863	75.52	22.36
3	Peroxide	10 min	2304526	76.41	24.63
4	Reduction	10 min	2302546	77.32	23.25
5	Thermal	10 min	2305632	78.24	22.63
6	Photolytic	10 min	2304215	78.15	21.95
7	Hydrolysis	10 min	2304563	74.63	26.15

Fig. 16: Standard Acid Degradation chromatogram

Fig. 17: Standard Alkali Degradation chromatogram

Fig. 18: Standard Peroxide Degradation chromatogram

Fig. 19: Standard Reduction Degradation chromatogram

Fig. 20: Standard Thermal Degradation chromatogram

Fig. 21: Standard Photolytic Degradation chromatogram

Fig. 22: Typical chromatogram of Hydrolysis Degradation

Table 12: Studies of Degradation Data from Tofisopam

S. No	Degradation Parameters	Time	Peak Area	% Recovery	% Degradation
1	Acid	10 min	2306402	78.20	21.25
2	Alkali	10 min	2304863	75.52	22.36
3	Peroxide	10 min	2304526	76.41	24.63
4	Reduction	10 min	2302546	77.32	23.25
5	Thermal	10 min	2305632	78.24	22.63
6	Photolytic	10 min	2304215	78.15	21.95
7	Hydrolysis	10 min	2304563	74.63	26.15

Fig. 23: Standard Acid Degradation chromatogram

Fig. 24: Standard Alkali Degradation chromatogram

Fig. 25: Standard Peroxide Degradation chromatogram

Fig. 26: Standard Reduction Degradation chromatogram

Fig. 27: Standard Thermal Degradation chromatogram

Fig. 28: Standard Photolytic Degradation chromatogram

Fig. 29: Standard Hydrolysis Degradation chromatogram

CONCLUSION:

In conclusion, a validated RP-HPLC method for determining the bulk and tablet dosage forms of L-Methyl Folate and Tofisopam has been established. The findings show that the technique was found to be specific, simple, precise, reliable and sensitive. For the determination of L-Methyl Folate and Tofisopam tablet dosage type, the method was successfully applied. For the quantitative calculation of L-Methyl Folate and Tofisopam separately and in conjunction with other drugs, many analytical procedures have been suggested thus, an attempt was made to develop and validate a reverse-phase high-performance liquid chromatographic method for the quality control of L-Methyl Folate and Tofisopam in pharmaceutical preparations with lower solvent consumption, together with the short analytical run time that leads to an environmentally friendly chromatographic procedure and will allow a large number of samples to be analyzed in a short period of time.

ACKNOWLEDGEMENTS:

The first authors wish to thank the organization of Icon Laboratories Ltd., Vijayawada, for allowing this work to be published. Support extended by all the colleagues of chemistry department in Bapatla Engineering College, Bapatla is thankfully acknowledged.

CONFLICTS OF INTERESTS:

The authors declare that there is no conflict of interest between them.

REFER ENCES:

1. Kelly CB, Mc Donnell, AP, Johnston TG, Mulholland C, Stephen JC, Dorothy Mc, Alun E, Alexdander SW. The MTHFR C677T polymorphism is associated with depressive episodes in patients from Northern Ireland. Journal of Psyc., 2004; 18(4): 567- 571.

2. Gilbody S, Lewis S, Lightfoot T. Methylenetetrahydrofolate reductase (MTHFR) genetic polymorphisms and psychiatric disorders: a HuGE review. Am. J. Epidemiol., 2007; 165(1): 1- 13.

3. Pietrzik K, Lynn B, Barry S. Folic acid and L-5-methyltetrahydrofolate: comparison of clinical pharmacokinetics and pharmacodynamics. Clinical Pharmacokinetics, 2010; 49(8):535-548.

4. L-MethylFolate monograph (Online) available on URL: https://www.drugbank.ca/drugs/DB04789

5. Skoog DA, Holler FJ, Nieman TA. Principles of Instrumental Analysis.5th ed. Newyork: Thomson Brooks/Cole; 1998; 329-35.

6. Willard HH, Merritt LL, Dean JA, Settle FA. Instrumental Methods Analysis.7th ed. New Delhi: CBS Publishers and Distributors; 1999; 1-12.

7. Sharma BK. Instrumental methods of chemical Analysis. 19th ed. Meerut: Geol Publishing house; 2003; 5-11.

8. Niraj V, Sangita P. Development and Validation of RP-HPLC Method for Simultaneous Estimation of Nebivolol and Indapamide in Pharmaceutical Dosage Form. Asian J. Pharm. Ana. 2014; 4(3): 98-102.

9. Govindarao K, Sowjanya V, Haripriya M. Method Development and Validation for Simultaneous Estimation of Omeprazole and Domperidone by RP-HPLC. Asian J. Pharm. Ana. 2015; 5(4): 195-205.

10. Kumaraswamy G, Lalitha R, Sadhana B, Suthakaran R, Pallavi K. Development and Validation of RP-HPLC Method for Simultaneous Estimation of Albendazole and Praziqantel in Tablet Dosage Form. Asian J. Pharm. Ana. 2015; 5(3): 115-118.

11. Janaki Pathi P, Appala Raju N. The Estimation of Epalrestat in Tablet Dosage Form by RP-HPLC. Asian J. Pharm. Ana. 2012; 2(2): 49-51.

12. Renu S, Badri Prakash N, Mahendra Kumar N, Joytosh B. Development and Validation of Simultaneous Estimation Method for Amoxycillin Trihydrate and Tinidazole in Tablet Dosage Form by RP-HPLC. Asian J. Pharm. Ana. 2013; 3(2): 66-71.

13. Kumaraswamy G, Ravindra N, Jyothsna B. Development and Validation of a Reversed-Phase HPLC Method for Simultaneous Determination of Aspirin, Atenolol and Amlodipine in Capsules Dosage Forms. Asian J. Pharm. Ana. 2014; 4(3): 116-120.

14. Akhilesh G, Swati R, Arun P. Method Development and Photolytic Degradation Study of Doxofylline by RP-HPLC and LC-MS/MS. Asian J. Pharm. Ana. 2011; 1(2): 29-33.

15. Vijaya Sri K, Anusha M, Ravinder Reddy S. A Rapid RP-HPLC Method development and Validation for the Analysis of Linagliptinin Bulk and Pharmaceutical Dosage Form. Asian J. Pharm. Ana. 2015; 5(1): 16-20.

16. Swapna J, Madhu CH, Srivani M, Sumalatha M, Nehalatha Y, Anusha Y. Analytical Method Development and Method Validation for the Simultaneous Estimation of Metformin hydrochloride and Pioglitazone hydrochloride in Tablet Dosage Form by RP-HPLC. Asian J. Pharm. Ana. 2012; 2(3): 85-89.

17. Thangabalan B, Avinash L, Chaitanya G, Sunitha N, Manohar Babu S. Stability Indicating RP–HPLC Method for the Estimation of Acamprosate in Pure and Tablet Dosage Form. Asian J. Pharm. Ana. 2013; 3(4): 141-146.

Received on 24.09.2020 Modified on 31.10.2020

Asian J. Research Chem. 2021; 14(1):28-36.

DOI: 10.5958/0974-4150.2021.00005.5