Method Development and Validation for the estimation of Class-2 Residual Solvents in Valacyclovir by HS-GC

 

Ramya Kuber B¹*, J. Soundarya²

¹Department of Pharmacognosy, Institute of Pharmaceutical Technology,

Sri Padmavati Mahila Visvavidyalayam (Women’s university), Tirupati, 517502, Andhra Pradesh, India.

²Department of Pharmaceutical analysis, Institute of Pharmaceutical Technology,

Sri Padmavati Mahila Visvavidyalayam (Women’s university), Tirupati, 517502, Andhra Pradesh, India.

 

ABSTRACT:

A simple headspace gas chromatographic (HS-GC) is a novel approach for the simultaneous detection of residual solvents such as ethanol has been devised and validated. Valacyclovir in ethanol, acetone, isopropyl alcohol, toluene, and acetic acid utilizing nitrogen as the carrier gas at a 4 mL/min flow rate with DB-620 column (80mx0.22mm) 1.8m inner diameter by using a flame ionization detector (FID). The headspace gas chromatographic technique provides an acceptable symmetry and resolution for all these residual solvents. The suggested method was shown to be satisfactory for determining the residual solvents of five distinct types. The method's validation findings showed that it is specific, exact, accurate, rugged, linear, and robust, with recoveries ranging from 80-120%. The %RSD (Relative Standard Deviation) for six injections should be NMT-10%. The correlation coefficient R² ≥ 0.999.

 

 

INTRODUCTION:

Residual solvents are evaporative organic compounds that are employed or present in various pharmaceutical products such as drug components, excipients, and drug products used in the synthesis of API. As a result, residual solvent testing should be carried out. When such solvents are known to be present as a result of a manufacturing or purifying operation.

They were classified into one of three categories based on their potential harm to human wellness:

Class 1 solvents are those that should be avoided.

Class-2 solvents: Solvents should be limited. 

Class 3 Solvents: Solvents have a low hazardous potential.

 

As a result, residual solvent detection in gas chromatography is possible¹.It is the commonly used method for the separation and analysis of volatile compounds in the world. Tswett invented the term "chromatography" in general. Tswett, Martin, Synge, and James were instrumental in the invention of the GC instrument².

 

Headspace separation is a process that involves injecting volatile material into gas chromatography for examination and separation. The compound under test in the headspace is determined not only by its volatility but also by the sample phase's affinity. This is known as the partition coefficient, and it is defined as the ratio of molecule concentration between two phases. Temperature, pressure, sample matrix, and equilibration time 3 all have a significant impact on the headspace approach³.

 

L-valine-2-[(2-amino-1, 6-dihydro-6-oxo-9-H-purin-9-yl) methoxy]valacyclovir The L-valyl ester prodrug of the antiviral drug acyclovir, ethyl ester, has an action against herpes simplex virus types 1 (HSV–1) and 2 (HSV–2), as well as varicella-zoster virus. 4 When valacyclovir is taken orally, it is swiftly transformed to acyclovir, which hinders the duplication of viral DNA after being further converted to the nucleotide analogue acyclovir triphosphate by viral thymidine kinase, cellular guanyl cyclase, and a variety of other cellular enzymes. Acyclovir triphosphate obstructs viral DNA polymerase competitively. Valacyclovir is quickly transformed to acyclovir, which is then phosphorylated to form acyclovir triphosphate. The inclusion of acyclovir triphosphate into the viral DNA growth chain leads to chain termination^4-7.

 

According to a literature review, many analytical techniques for valacyclovir estimation were discovered using RP-HPLC^8,9, LC-MS/MS¹⁰, and UV¹¹, methodologies. The purpose of this study was to identify the residual solvents, primarily ethanol, acetone, isopropyl alcohol, toluene, and acetic acid, in Valacyclovir pure medication.

 

DRUG PROFILE:

Name: VALACYCLOVIR

Structure:

 

Figure 1: Structure of Valacyclovir

 

Molecular Formula: C₁₃H₂₀N₆O₄

Molecular Weight: 324.3357

Category: Anti-Viral Drug

 

MATERIALS AND METHODS:

Chemicals and Reagents:

Valacyclovir is provided as a free sample by Chandra Laboratories located at Hyderabad. Qualigens provided ethanol (HPLC grade). Sigma Aldrich provided acetone, isopropyl alcohol, toluene, and acetic acid, while Qualigens provided dimethyl sulfoxide (DMSO).

 

Instruments:

The study was carried out using an Agilent gas chromatography model no. 7697A headspace sampler with the DB-620 column and Flame Ionization Detector using N₂ as a carrier gas.

Tables 1 and 2 provide the headspace and chromatographic conditions.

 

Table 1: Chromatographic conditions

 

Table 2: Headspace conditions

 

Preparation of solutions:

Preparation of Diluent:

As a diluent, dimethyl sulfoxide (DMSO) has been utilized.

 

Preparation of Blank:

Fill the headspace container with 1.0 mL of diluent and seal it immediately.

 

Standard Stock-I Preparation:

Take precisely 500mg of Ethanol, 500mg of Acetone, 500mg of Isopropyl alcohol, 500mg of Toulene, and 500 mg of Acetic acid in a 250ml volumetric flask holding 180ml of diluent, build up with diluent, and mix thoroughly.

 

Standard Stock-II Preparation:

Transfer 10ml of the aforementioned solution into a 200ml volumetric flask consisting of around 20mL diluent with the help of a pipette, then top up with diluent to volume. collect about 1ml of the above-mentioned solution by using pipette into a headspace vial and seal it.

 

Test Sample Preparation:

Transfer 500mg of the sample under examination (Valacyclovir API) to a 50mL volumetric flask, then introduce 35mL of the diluent, and vortex for about 5 min. Then add diluent to make up the difference in volume and thoroughly mix.

 

Fill a headspace vial with 1 mL of the solution that was prepared above and seal it.

 

Procedure:

The solutions that are prepared were transferred to a 5ml headspace vial and close it with a crimper. Headspace analysis is performed on the prepared standard and sample solutions.

 

Analytical Method Development:

For the development and simultaneous measurement of residual solvents in valacyclovir, several trials are carried out. Finally, as seen in Fig. 2, better resolution and peak form resulted in better separation.

 

RESULTS AND DISCUSSION:

Method Validation:

According to ICH guidelines, the process was verified for Linearity, System applicability, Precision, Robustness, specificity, Ruggedness, accuracy, LOD, and LOQ.

 

Linearity:

The studies on linearity for all the residual solvents are within the range of 50 to 150ppm for ethanol, acetone, isopropyl alcohol, toluene, and acetic acid (r2 >0.999) for the quantity of solvent determined by the given methods. Table 3 shows a summary of the findings.

 

Specificity:

The non-interference of residual solvents was used to determine specificity. The initial blank reagent was introduced into the headspace to acquire the peak. The graph was then recorded using a reference solution of residual solvents. Finally, the spiked sample is introduced into chromatograms to obtain the chromatogram. Figures 3, 4, and 5 show the chromatograms of the blank, reference, and spiked sample solutions, respectively. Table 4 shows the results of the specificity test.

 

 

 

Fig. 2: Optimized chromatogram for residual solvents

 

 

FIG. 3: Blank

 

 

FIG. 4: Standard

 

FIG. 5:  Spiked sample

 

 

Table 3: Data of linearity for residual solvents

 

Table.4 Specificity data of solvents:

 

System Suitability:

Six injections were used to test the system's suitability. Each solvent's percent RSD responses were found to be less than 10%. As a result, the system is appropriate for performing the analysis for estimating residual solvents in Valacyclovir.

 

Precision:

Method Precision:

The precision of the method was verified, and the percent Relative standard deviation for Ethanol, 0.05 for Acetone, 0.05 for Isopropyl alcohol, 0.05 for Toulene, and 0.05 for Acetic acid were found to be within limits. The precision percent RSD was also discovered to be NMT 10%.

 

Accuracy:

Ethanol, Acetone, Isopropyl alcohol, Acetone Toulene, and Acetic acid had typical recoveries of 80-120 percent, with an RSD of less than 10%.

 

Robustness:

The method's robustness was tested by varying the flow rate, and the parameters were determined to be within acceptable bounds; the percent RSD was less than 2%. As a result, the procedure is reliable.

 

Ruggedness:

The method's robustness was tested by monitoring multiple analysts, and the results that were determined are in accordance with the established parameters; the percent Relative standard deviation was less than 2%. As a result, the approach is durable.

 

LOD and LOQ:

Ethanol had a detection limit of 0.000084, Acetone had a detection limit of 0.00054, Isopropyl alcohol had a detection limit of 0.00018, Toulene had a detection limit of 0.000096 and Acetic acid had a detection limit of 0.000074.

 

Ethanol had a quantitative limit of 0.0025, Acetone had a limit of 0.00017, Isopropyl alcohol had a limit of 0.0005, Toulene had a limit of 0.00029, and Acetic acid had a limit of 0.0002.

 

CONCLUSION:

Based on the outcomes and specifications of the experiments, it was established that this newly created approach for estimating residual solvents of Ethanol, Acetone, Isopropyl alcohol, Toluene, and Acetic acid in Valacyclovir API was simple, specific, accurate, precise, robust, rugged, and of high resolution, and this procedure is more acceptable and cost-effective because of the shorter retention time and that it may be used efficiently at research institutes for regular analysis.

 

ACKNOWLEDGEMENT:

The authors are grateful to the professors at Sri Padmavathi Mahila Visvavidyalayam, IPT for their technical assistance and discussions.

 

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Accepted on 23.06.2022           © RJPT All right reserved

Research J. Pharm. and Tech 2022; 15(12):5388-5392.