Development and Validation of Head-Space Gas Chromatographic Method in Tandem with Flame ionized detection for the determination of Residual Solvents in Letermovir API synthesis

P. Bala Krishnaiah¹*, R.E.M. Prema Chandrika²

¹Vignan Institute of Pharmaceutical Technology, Beside VSEZ, Kapujaggarajupeta, Duvvada, Visakhapatnam-530049

²K.L.E.F, Vaddeswaram, Guntur District-522502, Andhra Pradesh.

*Corresponding Author E-mail: balakrishnaiahp@gmail.com

ABSTRACT:

Residual solvent testing is an integral part of reference material certification. A gas chromatography / flame ionization detector/headspace method has been developed and validated to detect and quantitate commonly used residual solvents in our production processes: ethyl acetate, MTBE, Toluene and isopropyl acetatein Letermovir API. HS-GC method in a simple and selective manner is delineated for the quantification and determination of Residual Solvents in Letermovir API. The separation of solvents along with drug on Chromatographic chamber was processed on USP G43 equivalent capillary column Thermo Scientific™ Trace GOLD™ TG-624 SilMS, 30 m × 0.32mm × 1.8µm column (P/N 26059-3390) using nitrogen as carrier gas by using different temperature gradient of FID Detectors. Linearity was observed in the range 10-50µg/ml for ethyl acetate, MTBE, Toluene and isopropyl acetate (r2>0.999) for the amount of solvent determined through sophisticated methods was in good agreement. The proposed methods were validated. The method of accuracy was assessed by recovery studies at three different levels.The method was found to be precise as indicated by the repeatability analysis, showing %RSD less than 10 for ethyl acetate, MTBE, Toluene and isopropyl acetate. All statistical data proves validity of the methods and can be used for routine analysis of pharmaceutical active ingredients for estimation of Residual Solvents of ethyl acetate, MTBE, Toluene and isopropyl acetate in Letermovir. Method validation comprised the following parameters: limit of detection (LOD), limit of quantitation (LOQ), linearity and range, accuracy, precision (repeatability and intermediate precision), system suitability, specificity, and robustness. Linearity (micrograms/mL) and LOQ (ppm) are listed for each solvent in manuscript. The present method was proven to be robust and accurate for quantitative analysis of residual solvent in neat materials.

KEYWORDS: Letermovir, Head-Space GC, Residual solvents.

INTRODUCTION:

Letermovir, an inhibitor of the CMV DNA terminase complex, and is administered orally or by intravenous infusion^1-3. Letermovir is meant to prevent the infective mechanism developed by cytomegalovirus (CMV) infection and also disease caused in association of an allogeneic hematopoietic stem cell transplant (HSCT) from CMV seropositive recipients [R+] in adults⁴. Molecular formula for Letermovir is designated as C₂₉H₂₈F₄N₄O₄ and a molecular weight of 572.55.

The IUPAC name of letermovir is (4S)-2-{8-Fluoro-2-[4-(3-methoxyphenyl) piperazin-1-yl]-3-[2-methoxy-5(trifluoromethyl) phenyl]-3,4-dihydroquinazolin-4-yl} acetic acid. Letermovir is very slightly soluble in water. It is available as 240mg and 480mg tablets. Residual solvent impurities in pharmaceuticals are trace amounts of volatile organic compounds used in drug production that remain in the final drug product⁵. One of the greatest demand for the pharmaceutical industry is maintaining the high standard quality of API and form of API in formulation from devoid of residual solvents to meet pharmacopoeia levels⁶. Furthermore, it is difficult and a challenging task for the separation and quantification of residual solvents of polar nature from pharmaceutical preparations as chemical synthesis is a process of involvement of water or polar solvents in the reaction^7-8 Residual solvents may also arise from exposure to packaging. Selectivity of Organic solvents during the process of manufacturing the pharmaceutical products is a key role in yield and determine of compounds with the characteristics like purity and solubility of crystalline form,. The effect of residual solvents and their quantities on synthesis and formulation of various drugs like Bendamustine Hydrochloride, rosuvastatin and atorvastatin, Telmisartan and solid dosage form by head space analysis of Gas chromatographic method are reported with the usage of different detectors^9-12. Rapid and specific method of Head space gas chromatographic (HSGC) technique for the estimation of residual solvents in bulk drug(Letermovir) was developed with proper validation procedures in this article. The HS-GC method is a quantification of the Residual Solvents in Letermovir API.

Instrument:

The Tri-Plus 500 Headspace auto-sampler was coupled to a Thermo Scientific™ TRACE™ 1310 Gas Chromatograph equipped with a Thermo Scientific™ Instant Connect Split/Split less (SSL) Injector and a Thermo Scientific™ Instant Connect Flame Ionization Detector (FID). separation of components through Chromatographic method was eluted on a USP G43 equivalent capillary column Thermo Scientific™ Trace GOLD™ TG-624 SilMS, 30m × 0.32mm × 1.8µm column (P/N 26059-3390) using nitrogen as carrier gas. The complete operational conditions are given in Table 1. An analytical balance (XS 205 from Mettler Toledo) and auto-pipette (100 – 1000µL from Eppendorf) were used. The headspace injector and GC conditions are provided in Table I.

Chemicals and Reagents:

Used Chemicals were obtained from the following suppliers: methanol (sigma-aldrich, Mumbai, India), methyl-tert-butyl ether (MTBE), Toluene (PhMe), Isopropyl Acetate (IA), and Ethyl Acetate (Qualigens, Mumbai, India). Dimethyl slufoxide (DMSO) HPLC grade (Spectrochem, Mumbai, India). Letermovir API was obtained from Silpamedicare, Mumbai, India.

Synthesis of Letermovir:

It involved in series of seven steps under concise asymmetric synthesis from commercially available materials^13-15. Key to the eﬀectiveness of this process is a novel cinchonidine-based PTC-catalyzed aza-Michael reaction to conﬁgure the single stereocenter¹⁶ is shown in schematic figure-1. The residual solvents estimated in the present research are also listed in schematic figure-1.

Chromatographic Conditions:

A volume of 1ml standard and sample solution was injected into the GC injection port maintained at a temperature of 170°C which was under a split ratio of 1:10. Mobility of nitrogen gas. was carried through the coloumn at a pressure of 14 psi with a flow of one mL per minute. The detector temperature was maintained at 250°C. Temperature gradient was initialized at 40°C for a period of 12 min, which was again raised in the temperature at a rate of 10°C min-1 up to 220°C for 5 min.

Table 1. HS-GC-FID analytical parameters for Class 2A residual solvent screening according to the proposed method modifications.

TRACE 1310 GC System Parameters
Inlet Module, Mode:	SSL, split
Split Ratio:	10: 1
Septum Purge Mode, Flow:	Constant, 5mL/min
Carrier Gas, Carrier Mode, Flow-	N2 , constant flow, 2.5 mL/min
Oven Temperature Program:
Temperature-1:	40°C
Hold Time:	1 min
Temperature-2:	170°C
Rate-	20°C/min
FID-Parameters:
Temperature	250°C
Air Flow	400mL/min
H₂ Flow	40mL/min
N2 Flow	40mL/min
Acquisition Rate	25 Hz
Tri-Plus 500 HS Auto-sampler Parameters:
Incubation Temperature:	80°C
Incubation Time:	20min
Vial Shaking:	Fast
Vial Pressurization Mode:	Pressure
Vial Pressure (Auxiliary Gas Nitrogen)	130 kPa
Vial Pressure Equilibration Time	1min
Loop size	1mL
Loop/Sample Path Temperature	80°C
Loop Filling Pressure	73 kPa
Loop Equilibration Time	1 min
Needle Purge Flow Level	2
Injection Mode	Standard
Injection Time	1 min

Standard and stock solutions:

Standard solution of the four residual solvents present in Letermovir was prepared in the following manner: Transfer 10mg of each of methyl-tert-butyl ether (MTBE), Toluene (PhMe), Isopropyl Acetate (IA), and Ethyl Acetate working standards into a 100ml volumetric flask, dissolve and dilute with Dimethyl Sulfoxide (DMSO) as diluent. The resulting solution contains 100μg/mL of each of methyl-tert-butyl ether (MTBE), Toluene (PhMe), Isopropyl Acetate (IA), and Ethyl Acetate standard solutions. The prepared stock solutions were stored at 4°C and protected from light.

Solutions for validation study:

Calibration and Quality control samples:

Calibration standards (10–50μg/mL methyl-tert-butyl ether (MTBE), Toluene (PhMe), Isopropyl Acetate (IA), and Ethyl Acetate were prepared from standard stock solutions by appropriate dilution with Dimethyl sulfoxide (DMSO) as diluent. Quality control (QC) samples are prepared at three concentrations of the linearity range (30μg/mL, 40μg/mL and 50μg/mL each for methyl-tert-butyl ether (MTBE), Toluene (PhMe), Isopropyl Acetate (IA), and Ethyl Acetate were prepared from the standard solutions.

Preparation of the Sample solution:

Accurately weighed 100mg of Letermovir bulk drug was extracted with 100ml of diluent and the filterate (5ml) was diluted to a volume upto 50ml with the DMSO as diluent.

Method Validation:

The developed chromatographic method was validated for selectivity, linearity, precision, accuracy, sensitivity, robustness and system suitability.

Specificity:

The Letermovir API sample was spiked with methyl-tert-butyl ether (MTBE), Toluene (PhMe), Isopropyl Acetate (IA), and Ethyl Acetate individually and each sample was chromatographed to examine interference, if any, of the residual solvent peaks with each other. The retention time for standard methyl-tert-butyl ether (MTBE), Toluene (PhMe), Isopropyl Acetate (IA), and Ethyl Acetate was found to be 2.32, 5.044, 5.81, 8.19respectively and DMSO as diluent is eluted at 11.5 minutes.

System suitability:

The system suitability was assessed by six replicate analyses of the residual solvents at concentrations of 40 μg/ mL each of methyl-tert-butyl ether (MTBE), Toluene (PhMe), Isopropyl Acetate (IA), and Ethyl Acetate.

The acceptance criterion was ±2% for the RSD for the peak area and retention times for all four residual solvents. The criterion for system suitability was that the resolution between the above-mentioned critical pair should not be less than 1.5 and it was found to be well above the minimum passing limit.

Linearity:

Linearity of the method was evaluated at four equi-spaced concentration levels by diluting the standard solutions to give solutions over the ranges 40–120% target concentration for all four residual solvents respectively. The calibration curves were constructed at four concentrations between (10–50 μg/ mL for each of methyl-tert-butyl ether (MTBE), Toluene (PhMe), Isopropyl Acetate (IA), and Ethyl Acetate. The peak areas of the injected samples in triplicate manner was imported in Microsoft Excel® format for design of calibration curves. Least square regression method was applied for evaluation of linearity through linear regression analysis.

Table-2: Linearity data of Residual solvents present in Letermovir API.

Linearity range	MTBE	Ethyl acetate	Isopropyl Acetate	Toluene
10 μg/ mL	285.75	1351.01	10722.25	1490.1
20 μg/ mL	525.42	2665.46	23115.3	2915.45
30 μg/ mL	775.21	3882.06	35431.25	4335.47
40 μg/ mL	1010.52	5193.21	48825.78	5752.87
50 μg/ mL	1275.87	6635	62316.38	7213.24
Corr.Coeff.	0.9997	0.9992	0.9995	0.9998

Table-5: Recovery Study of working dilutions of Residual solvents present in Letermovir API

Parameter	MTBE	Ethyl acetate	Isopropyl Acetate	Toluene
80% Accuracy	113.5	116.9	115.6	116.2
100% Accuracy	100.7	99.9	97.4	99.4
120% Accuracy	105.4	108.5	100.7	107.7

Sensitivity:

Limits of detection (LOD) and quantification (LOQ) were estimated from the regression equation method. The limit of detection was determined, by injecting progressively low concentrations of four analytes of interest. The quantification limit was determined as the lowest concentration level that provided a peak area with s/n ratio is 10.

Table-6: LOD and LOQ data for the four Residual solvents present in Letermovir-API.

Parameter	MTBE	Ethyl acetate	Isopropyl Acetate	Toluene
LOD	5 μg/mL	2 μg/mL	0.05 μg/mL	1 μg/mL
LOQ	15 μg/mL	6 μg/mL	1.15 μg/mL	3 μg/mL

CONCLUSION:

In summary, a headspace gas chromatographic method was established and validated for the determination of four residual solvents in Letermovir. The established method shows high sensitivity, good accuracy and linearity, which was applied successfully in the quality control of three batches of Letermovir. The residual solvents methanol, ethanol, acetone and toluene were determined. The developed method is specific, accurate, precise and rugged as per ICH guidelines.

REFERENCES:

1. Manfred M, Thomas S, Andreas U, Steffen W, Helga R-Schaeff, Holger Z, Peter L. In vitro evaluation of the activities of the novel anticytomegalovirus compound AIC246 (letermovir) against herpesviruses and other human pathogenic viruses. Antimicrob. Agents Chemother. 2012;56(2):1135-37.

2. Thomas G, Guy H, Nicole E, Helga R-Schaeff, Holger Z, Peter L. The novel anticytomegalovirus compound AIC246 (Letermovir) inhibits human cytomegalovirus replication through a specific antiviral mechanism that involves the viral terminase. J. Virol. 2011;85(20):10884-93.

3. Peter L, Guy H, Tobias W, Judith B, Daniela P, Thomas G, Helga R-Schaeff, Holger Z. In vitro and in vivo activities of the novel anticytomegalovirus compound AIC246. Antimicrob. Agents. Chemother. 2010;54(3):1290-97.

4. Steffen W, Holger Z, Peter L. In vitro drug combination studies of Letermovir (AIC246, MK-8228) with approved anti-human cytomegalovirus (HCMV) and anti-HIV compounds in inhibition of HCMV and HIV replication. Antimicrob. Agents. Chemother. 2015;59(6):3140-48.

5. International Conference on Harmonization (ICH) of Technical Requirements for the Registration of Pharmaceuticals for Human Use, Q3C (R4): Impurities: guideline for residual solvents, Step 4, July 1997.

6. (2) International Conference on Harmonization, Guidance on impurities: residual solvents. Fed. Regist. 62: 67377-88, 1997.

7. (3) USP <467>, General Chapter on Organic volatile impurities, United States Pharmacopeia, (USP 32-NF 27). Pharmacopoeia Convention Inc., Rockville, Maryland, August 2009.

8. (4) Residual Solvents (5.4), European Pharmacopoeia, Supplement 4.6, Directorate for the Quality of Medicines of the Council of Europe, Strasbourg, fourth edition, 2004, 3911.

9. Ramesh Babu J, Suhasini J, Vidyadhara S. Residual Solvents in Bendamustine Hydrochloride By Headspace Chromatography. Asian J. Pharm. Ana. 2018; 8(1):07-12.

10. Ali Ismail, Youssef Alahmad. Determination of ethanol and n-hexane residues in bulk rosuvastatin and atorvastatin and their dosage forms using HS-GC-MS developed method. Research J. Pharm. and Tech 2018; 11(11): 4829-4836.

11. Tentu NageswaraRao, T.B. Patrudu, Suneel Kumar. A, N. Krishna Rao, Karri Apparao. A New Analytical Method Validation and Quantification of Residual Solvents in Telmisartan Bulk Drug Product by Headspace gas Chromatographic Method. Research J. Science and Tech. 2018; 10(2):98-104.

12. Shoeb Alahmad, Mhd.Amer Almardini, Mahzia yahia. Validated HS-GC-FID Method for Determination of Residual Ethanol in. Solid Dosage Form. Research J. Pharm. and Tech. Feb. 2014; 7(2):184-187.

13. Ahrendt KA, Borths CJ, MacMillan DWC. New strategies for organic catalysis: the ﬁrst highly enantioselective organocatalyticDiels−Alder reaction. J. Am.Chem. Soc. 2000;122:4243–4.

14. List B, Lerner RA, Barbas III CF. Proline-catalyzed direct asymmetric aldol reactions. J. Am.Chem.Soc.2000;122:2395–6.

15. MacMillan DWC. The advent and development of organocatalysis. Nature. 2008;455:304–8.

16. Marqués-López E, Herrera RP, Christmann M. Asymmetric organocatalysis in total synthesis – a trial by ﬁre. Nat.Prod.Rep. 2010;27:1138–67.

Received on 12.12.2020 Modified on 03.04.2021

Research J. Pharm.and Tech 2022; 15(3):1023-1028.

DOI: 10.52711/0974-360X.2022.00171