New LC-MS Method Development and Validation for Quantitative Estimation of 2-Acetoxy Ethyl Acetoxy Methyl Ether in Acyclovir

 

Indhu Priya Mabbu^1,2*, G. Sumathi³, N. Devanna¹

¹Research Scholar, Department of Chemistry, Jawaharlal Nehru Technological University Anantapur,

College of Engineering, Anantapur-515002, Andhra Pradesh.

²Department of Chemistry, S.V.College of Engineering, Tirupati-517507.

³Department of Chemistry, Anantha Lakshmi Institute of Technology and Sciences,

Anantapur-515002, Andhra Pradesh.

 

ABSTRACT:

The objective of the proposed method is to develop and validate a specific, precise and accurate LC-MS method for the determination of 2-acetoxy ethyl acetoxy methyl ether (AEM) in Acyclovir. An isocratic separation was done by using YMC-PACK PRO C18, (150 x 4.6 mm, 3µm) column with a mobile phase composition of Buffer (0.1% Ammonia in water) : Methanol(25 :75v/v)at a flow rate of 0.4mL/min, diluent solution of 0.2% of 0.1N NaOH in Methanol were monitored on a triple quadrupole mass spectrometer, operating in the multiple reaction monitoring (MRM) Acquisition mode and possitive polarity mode. The Retention time of AEM was found to be 5.344 minutes, LOD and LOQ were observed at 0.03 ppm and 0.11 ppm concentration respectively by signal to noise ratio method, linearity was found in the concentration range of 0.18-0.55 ppm with correlation coefficient of 0.996 and accuracy in the range of 97.7- 101% was obtained by performing percentage recovery studies. The % RSD was observed to be less than 10% for six replicates said to be precise. The proposed method was validated for accuracy, precision, sensitivity, and linearity successfully employed for quantitative determination of AEM in acyclovir.

 

 

INTRODUCTION:

The anti viral drug Acyclovir is a nucleic acid analogue of guanosine, mainly used in the treatment of herpes simplex virus infections^1-2. During the synthesis, formulation, manufacturing and storage of any drug there is a chances of presence of foreign substances, process impurities, unreacted portion of intermediate substances and related substances. Identification and quantification of those potential impurities and related substances in the drug substance and drug product in essential to get FDA approval and to produce a highly purified drug.

 

2-acetoxy ethyl acetoxy methyl ether (AME)) is used as intermediate for the synthesis of acyclovir hence there is a chance for AME to exist as potential impurity in the acyclovir drug substance. Figure No: 1

 

Figure No. 1: Structure of AEM and Acyclovir

 

Extensive literature search reports that, various methods are available for the estimation of acyclovir alone and in combination with other drugs, using UV spectroscopic methods and stability indicating RP- HPLC method^2-7. Few LC methods for simultaneous estimation of acyclovir and related impurities^8-9. Bio analytical RP- HPLC and LC-MS methods to estimate acyclovir were reported^10-12. Till date no method has reported to estimate AEM in acyclovir by using LC-MS method; hence research has under taken to develop a simple, economical, sensitive, specific and accurate LC-MS method to estimate AEM in acyclovir.

 

MATERIALS AND METHODS:

The AEM reference standard (claim 99.28%) was provided by HETERO Drugs as gift sample, HPLC grade methanol was obtained from Finar Chemicals Limited, Ahmadabad, India. All the glass wares used in this research work were made of high quality borosilicate glass and the solvents and solutions used were filtered by using Millipore (0.45µm) filters.

 

LC-MS method conditions:

Method was performed by Agilent LC-MS with auto sampling system. The chromatographic separation was performed using YMC-PACK PRO C18, (150 x 4.6 mm, 3µm) column. Separation was successfully done by using a mobile phase composition of 0.1%ammonia buffer: methanol (25:75 v/v) at a flow rate of 0.4 ml/min with injection volume of 20µL. The pH of the mobile phase was 6.8. 0.2% of 0.1N NaOH in methanol used as diluents. The temperature maintained in auto sampler was 5°C and ambient temperature maintained in column. MS parameters were described in the Table-1.

 

Table 1: MS parameters

Ionization mode	ESI
Acquisition mode	MRM
Polarity Mode	Positive
Ch1	199.05 (M+Na)
Interface	4.5 KV
Detector	1.76 KV
DL temp.	300ºC
Heat Block	350ºC
Nebulizing Gas Flow	3.0 L/min.

 

Preparation of AEM Standard Solution:

Accurately weigh and transfer about 12.5mg of AEM standard in to 50mL volumetric flask dissolve and dilute to volume with diluent and mix well. Transfer 2.8mL into a 10mL volumetric flask dissolve and dilute to volume with diluent and mix well. Further transfer 0.25mL into a 25mL volumetric flask dissolve and dilute to volume with diluent and mix well to obtain concentration of 10ppm. Transfer 0.74mL of the above solution into a 20mL volumetric flask and dilute to volume with diluent and mix well to obtain 0.37ppm.

 

Preparation of Test Solution:

Accurately weigh and transfer about 350mg of the acyclovir test sample into a 15mL. Centrifuge tube add 5mL of diluent and dissolve by sonication, then Centrifuge at 4000rpm for 10 min and transfer into a 1.5mL sample vial.

 

Preparation of Test Sample Spiked with Limit Level (Standard) Solution:

2-Acetoxy ethyl acetoxy methyl ether (AEM)was spiked into Acyclovir, Spiked concentration was 0.37 ppm with respect to acyclovir sample solution.

 

Method validation:

Validation is establishing documented evidences, which provides a high degree of assurance that a specific process will consistently produce a product meeting its predetermined specifications and quality characteristics^13-17.

 

System suitability Test:

Test for system suitability was performed by injecting blank solution once and standard solution of AME concentration about 0.37ppm (100% level) for six times into LC-MS system. The system suitability was established by evaluating the system suitability parameters from the chromatograms thus obtained. Typical system suitability parameters include % RSD, number of theoretical plates

 

Linearity:

The linearity of an analytical method aims to elicit whether the test result are directly proportional to concentration¹⁸. Linearity study was conducted for AEM in the range of LOQ level (0.11 ppm) to about 150% of limit (0.55ppm) by injecting each concentration level six time and plotted a graph with average peak areas v_s Concentration to find out regression coefficient value.

 

Accuracy:

Percentage recovery method used to carry out accuracy of the method, at LOQ level and Limit level. A known amount of AEM spiked separately to pre-analyzed samples LOQ level and limit level, each spiked sample injected for three times and percent recoveries were calculated at each level¹⁹.

 

Precision:

System precision was determined by injecting AEM concentration of 0.11ppm (LOQ level) for six times in the same day (intra-day). Inter-day precision was determined by analyzing same AEM concentration level daily three times for three days and % RSD was calculated

 

Sensitivity (LOD and LOQ):

Limit of detection (LOD) is defined as the lowest amount of an analyte that can be detected but not quantified. Limit of quantification (LOQ) of an analytical method is the lowest amount of analyte that can be quantified with effective precision and accuracy ²⁰. LOD and LOQ values were established for AEM using signal to noise ratio. LOD solution was prepared so as to obtain the S/N ratio is about 3:1 to 5:1. Based on the concentration obtained from LOD, the LOQ solution was prepared (3 times to LOD concentration) so as to obtain the signal to noise ratio is about 10:1 to 15:1

 

RESULTS AND DISCSSION:

Initially, the solubility of AEM was checked in various solvents. It was found to be slightly soluble in methanol and water.

 

Method Optimization:

Optimization of the LC-MS conditions were carried out by performing several trials to obtain good retention time, peak symmetry and relative standard deviation within the limits and possible optimal. After several trails, a method using mobile phase composition of buffer(0.1% Ammonia in water) : methanol(25 :75v/v) at a flow rate of 0.4mL/min on YMC-PACK PRO C18, (150 x 4.6 mm, 3µm) column was found to be the most suitable and acceptable. Optimized method resulted in chromatogram with AEM eluting at 5.34min (Figure No: 2) and parent and base peak at 199 in mass spectrum (Figure No: 3).

 

Figure No. 2: Optimized chromatogram of AEM

 

Figure No. 3: Mass spectrum AEM

 

Method validation:

The proposed method was validated according to Q2 specifications of the ICH guidelines.

 

System Suitability:

The system suitability was established by assessing the system suitability parameters from the chromatograms thus obtained. Typical system suitability parameters include % RSD, Tailing factor (T) and Theoretical plates (N). All the parameters measured values (Table-2) were satisfying the acceptance criteria.

 

Table 2: Results of System Suitability Test for AME

Injection	Retention Time (min)	Peak area µV x sec
1	5.34	410963
2	5.32	418582
3	5.29	419885
4	5.36	417561
5	5.34	418280
6	5.28	412507
Mean		416296.3
Standard deviation		3645
%RSD		0.88

 

Linearity:

Linearity study was conducted for AEM in the range of LOQ level to about 150% of limit. Linearity graph is drawn for AEM in the range of LOQ to 150% of limit. Calibration curve was constructed by plotting area against concentration and regression equation was computed. r²value of linearity curve was 0.996, linearity graph and peak area values were shown in Figure No:4 and Table-3 respectively.

 

Table 3: Linearity of AEM

Percentage Level	Concentration (ppm)	Average Peak Area of AEM
Level (LOQ)	0.11	139115
50(limit)	0.18	195801
75	0.27	297144
100	0.37	418583
125	0.46	504267
150	0.55	593969
r2		0.996

 

Figure No. 4: Calibration curve of AEM

 

Accuracy:

The percentage recovery of AME from the spiked sample solutions were found to be in the range of 97.7 – 101% (Table-4) indicate that accuracy of proposed analytical method was within the acceptance criteria (±25%) of the ICH guidelines.

 

Table 4: Percentage recovery data for AME

Level	Concentration	Injection	Spiked sample peak areas	% Recovery
LOQ	0.11ppm	1	137693	97.7
		2	145645	103.5
		3	142256	101
Limit (50%)	0.18ppm	1	459055	111.4
		2	413417	100.6
		3	420206	102.3

 

Precision:

Variation in peak areas of the LOQ concentration (0.11ppm) within the same day (intra-day)and between days (inter-day) were analyzed,% RSD of peak area values of AME were found to 3.1 and 2.50for intra-day(Table-5) and inter-day precision respectively (Table-6).The low values of (≤10) these statistical parameters represents the method with good precision.

 

Table 5: Results of intraday Precision

LOQ Solution	Peak Area of AEM
Injection-1	145065
Injection-2	133165
Injection-3	141523
Injection-4	144187
Injection-5	142764
Injection-6	144513
AVERAGE	141869.5
STDEV	4455.5
%RSD	3.1

 

Table 6: Results of inter day Precision

Day	LOQ Solution	Peak Area of AEM
Day-1	Injection-1	145065
	Injection-2	144187
	Injection-3	141523
Day-2	Injection-1	133213
	Injection-2	142764
	Injection-3	143251
Day-3	Injection-1	143182
	Injection-2	144513
	Injection-3	141521
	AVERAGE	142135
	STDEV	3562
	%RSD	2.5

 

Sensitivity:

LOD and LOQ were found to be0.03ppm and 0.11ppm, which indicate that the method has good sensitivity. Table-7 represents the results of validation parameter with acceptance limit.

 

Table 7: Summary of the parameter values with acceptance limit

Parameters	Results	Acceptance Criteria
Specificity	AEM eluted at retention time of 5.3 min. There is no interference due to blank at the retention time of AEM	There should be no interference due to blank at the retention time of AEM
LOD	S/N ratio for AEM at LOD level is 3.5 (0.03 ppm).	The Signal to Noise ratio for AEM should be about 3:1 to 5:1.
LOQ	S/N ratio for AEM at LOQ level is 14.7 (0.11 ppm)	The Signal to Noise ratio for AEM should be about 10:1 to 15:1.
Precision	The % RSD for peak areas of AEM from six replicate injections is 3.1.	The % RSD for peak areas of AEM from six replicate injections should not be more than 10.0.
Linearity	Correlation coefficient value for AEM derived from the linearity graph is 0.996.	Correlation coefficient value of AEM derived from linearity graph should not be less than 0.99.
Accuracy	LOQ Level:
	The % recoveries for AEM at LOQ level is in the range of 97.7 to 103.5.	The % recoveries of AEM should be in between 80 to 120.
	Limit Level:
	The % recovery for AEM at limit level is in the range of 100.6 to 111.4.

 

CONCLUSION:

The simple mobile phase composition for the LC method was developed for study of AME in acyclovir in pure and pharmaceutical dosage form..The validated method is very good, sensitive, accurate, and precise. Moreover it has the benefit of short run time and the likelihood of analysis of more number of samples, both of which significantly reduce the analysis time per sample. Hence this method can be perfectly suitable for routine analysis of AME in acyclovir pure and dosage forms in quality control department in the pharmaceutical industry.

 

ACKNOWLEDGMENT:

The authors are thankful to Fortune Pharma, Hyderabad for providing lab facilities and chemicals. The authors are also thankful to Department of Chemistry, Jawaharlal Nehru Technological University Anantapur, Andhra Pradesh for encouragement.

 

AUTHORS’ CONTRIBUTIONS:

All the authors contributed equally in design and frame of the work, acquisition and interpretation of data and manuscript preparation, all authors have read the prepared manuscript and approved for the publication.

 

CONFLICT OF INTEREST:

No conflict of interest from all the authors.

 

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Accepted on 29.11.2020           © RJPT All right reserved

Research J. Pharm.and Tech 2021; 14(11):5881-5885.