Development and Validation of Head-space Gas Chromatographic Method in Tandem with Flame ionized detection for the determination of Residual solvents in Simeprevir API Synthesis
Department of Chemistry, Vel Tech Rangarajan Dr. Sagunthala R and D Institute of Science and Technology, Avadi, Chennai - 600062, India.
*Corresponding Author E-mail: mallibabua@gmail.com
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: Methanol, Tetrahydrofuran, Toluene, Dichloromethane and Dichloroethane in Simeprevir API. A simple and selective HS-GC method is described for the determination & quantification of Residual Solvents in Simeprevir API. Chromatographic separation was achieved on 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 by using different temperature gradient of FID Detectors. Linearity was observed in the range 40-120% of standard concentrations for Methanol, Tetrahydrofuran, Toluene, Dichloromethane and Dichloroethane (r2>0.999) for the amount of solvent estimated by the proposed methods was in good agreement. The proposed methods were validated. The accuracy of the methods was assessed by recovery studies at three different levels. Recovery experiments indicated the absence of interference from commonly encountered diluent and API. The method was found to be precise as indicated by the repeatability analysis, showing %RSD less than 10 for Methanol, Tetrahydrofuran, Toluene, Dichloromethane and Dichloroethane. 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 Methanol, Tetrahydrofuran, Toluene, Dichloromethane and Dichloroethane in Simeprevir. Baseline separation of all five solvents and Simeprevir API is achieved within 20.5 minutes of analysis time. 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 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: Simeprevir, Head-Space GC, Residual solvents, FID analysis and HS-GC method.
Simeprevir is an oral, direct acting hepatitis C virus (HCV) protease inhibitor that is used in combination with other antiviral agents in the treatment of chronic hepatitis C, genotypes 1 and 4. Simeprevir is an orally bioavailable inhibitor of the hepatitis C virus (HCV) protease complex comprised of non-structural protein 3 and 4A (NS3/NS4A), with activity against HCV genotype 1. Upon administration, simeprevir reversibly binds to the active center and binding site of the HCV NS3/NS4A protease and prevents NS3/NS4A protease-mediated polyprotein maturation. This disrupts both the processing of viral proteins and the formation of the viral replication complex, which inhibits viral replication in HCV genotype 1-infected host cells1-3. Simeprevir is an azamacrocycle and a lactam. The chemical name for simeprevir is (2R, 3aR,10Z,11aS,12aR,14aR) - N(cyclopropyl sulfonyl) -2-[[2-(4-isopropyl- 1,3-thiazol- 2-yl)-7-methoxy-8-methyl-4quinolinyl] oxy]-5-methyl - 4,14-dioxo-2,3,3a,4,5,6,7,8,9,11a,12,13,14,14a tetra deca hydro cyclopenta[c]cyclo propa[g][1,6]diaza cyclo tetra decine-12a(1H) carboxamide. Its molecular formula is C38H47N5O7S2 and its molecular weight is 749.94. Simeprevir drug substance is a white to almost white powder14. Simeprevir is practically insoluble in water over a wide pH range. It is practically insoluble in propylene glycol, very slightly soluble in ethanol, and slightly soluble in acetone. It is soluble in dichloromethane and freely soluble in some organic solvents (e.g., tetrahydrofuran and N, N- dimethylformamide)24. OLYSIO (simeprevir) for oral administration is available as 150 mg strength hard gelatin capsules. Each capsule contains 154.4mg of simeprevir sodium salt, which is equivalent to 150mg of simeprevir. OLYSIO® is available as a white gelatin capsule marked with “TMC435 150” in black ink. Each capsule contains 150mg simeprevir9. The determination of residual solvents in drug substances, excipients or drug products is known to be one of the most difficult and demanding analytical tasks in the pharmaceutical industry10. Furthermore, determination of the polar residual solvents in pharmaceutical preparations is still an analytical challenge mainly because these compounds are quite difficult to remove from water or polar solvents11-12. Residual solvents may also arise from exposure to packaging16. Organic solvents are widely used in the manufacturing process of pharmaceutical products. Their application in the synthesis of active pharmaceutical ingredients (APIs) is of exceptional importance as the use of appropriate solvents may enhance the yield or determine characteristics such as crystal form, purity and solubility13. In the current article we are reporting the development and validation of a rapid and specific Head space gas chromatographic (HSGC) method for the determination of organic volatile impurities (residual solvents) in Simeprevir bulk drug. The HS-GC method is a quantification of the Residual Solvents in Simeprevir API.
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). Chromatographic separation was achieved on a 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. For this application, nitrogen is a viable alternative to helium (which is expensive and prone to regional shortage). Nitrogen allows for highly efficient GC separations and can easily be produced in the lab at high purity by using a nitrogen generator and making it very cost effective. The complete operational conditions are given in Table 1. An analytical balance (XS 205 from Mettler Toledo) and auto-pipette (1mL, 2mL, 5mL and 10mL from Eppendorf) were used17. The headspace injector and GC conditions are provided in Table I.
Used Chemicals were obtained from the following suppliers: Methanol, Toluene (sigma-aldrich), Dichloromethane, Dichloroethane and Tetrahydrofuran (THF) (Qualigens). Dimethyl sulfoxide (DMSO) HPLC grade (S.D fine). Simeprevir API was obtained from Silpa medicare, Mumbai, India.
The development of a concise asymmetric synthesis of the antiviral development candidate Simeprevir is reported, proceeding in >60% yield over a total of seven steps from commercially available materials5-7. Key to the effectiveness of this process is a novel cinchonidine- based PTC-catalyzed aza-Michael4 reaction to configure the single stereocenter8 is shown in schematic figure-1. The residual solvents estimated in the present research are also listed in schematic figure-1.
Figure 1: Synthesis of Simeprevir
A volume of 1ml standard and sample solution was injected into the GC injection port. The temperature of the injection port was maintained at 170°C at a split ratio of 1:10, with nitrogen as a carrier gas. The pressure was maintained at 14 psi with flow of 1mL min-1. The temperature of the detector was set at 250°C. Temperature gradient was maintained at 40°C for five min and then increased at a rate of 20°C min-1 up to 230
°C to a final temperature of 230°C and maintained for 5 min15.
Standard solutions:
Standard solution
of the five residual solvents
present in Simeprevir was prepared in the following manner:
Weigh accurately about methanol 300mg, Toluene 89 mg, Dichloromethane 60mg, Dichloroethane 180mg and Tetrahydrofuran (THF) 70mg working
standards into a 100ml volumetric flask, dissolve and dilute with Dimethyl
Sulfoxide (DMSO) as diluent. Further dilute 10ml
of the above solution into 100mL volumetric flask and make up with DMSO. Take 5mL of the solution into HS vial
as standard solutions.
Table 1. HS-GC-FID analytical parameters for Class 2A residual solvent screening according to the proposed method modifications.
|
Nitrogen) |
|
|
· Vial Pressure Equilibration Time |
1 min |
|
· Loop size |
1 mL |
|
· 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 |
Accurately weighed 500mg of Simeprevir bulk drug and transferred in to a Head space vial. Add 5mL of diluent, dissolve and seal the vial tightly.
The developed chromatographic method was validated for selectivity, linearity, precision, accuracy, sensitivity, robustness and system suitability18.
The Simeprevir API sample was spiked with methanol, Toluene, Dichloromethane, Dichloroethane and Tetrahydrofuran (THF) individually and each sample was chromatographed to examine interference, if any, of the residual solvent peaks with each other. The retention time for standard solvents of methanol, Toluene, Dichloromethane, Dichloroethane and Tetrahydrofuran (THF) was found to be 2.95, 5.35, 7.75, 9.85, 13.0
respectively and DMSO as diluent is eluted at 16.5 minutes.
Figure-2: Specificity Chromatogram of Residual solvents present in Simeprevir
The system suitability was assessed by six replicate analyses of the residual solvents of standard solution for methanol, Toluene, Dichloromethane, Dichloroethane and Tetrahydrofuran (THF). The acceptance criterion was ±2% for the RSD for the peak area and retention times for all five residual solvents. The system suitability parameters with respect to theoretical plates, tailing factor, repeatability and resolution between diluent peak and peaks of the other residual solvents are accepted by the ICH guidelines. 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 limit23.
Linearity of the method was evaluated at five concentration levels by diluting the standard solutions to give solutions over the ranges 40–120% target concentration for all five residual solvents respectively. The calibration curves were constructed at five concentrations for each of Methanol, Toluene, Dichloromethane, Dichloroethane and Tetrahydrofuran (THF)20. These were injected in triplicate and the peak areas were inputted into a Microsoft Excel® spreadsheet program to plot calibration curves19. The linearity was evaluated by linear regression analysis, which was calculated by the least square regression method. The peak areas of the drugs to drugs concentration were used for plotting the linearity graph. The correlation coefficient (r2) values for all residual solvents were found to be higher than 0.999 and the calibration curves were linear within the range22. Table 2 shows the linearity values for the residual solvents
Table-2: Linearity data of Residual solvents present in Simeprevir API.
|
Concentration of Linearity Range for standard solution |
Methanol Area |
Toluene Area |
Dichloro- Methane Area |
Dichloro- Ethane Area |
Tetrahydro- furan (THF) Area |
|
40% concentration |
1998.85 |
4298.84 |
563.11 |
29075.71 |
3413.43 |
|
60% concentration |
4094.96 |
5981.91 |
788.76 |
57775.66 |
6845.35 |
|
80% concentration |
6111.18 |
7785.22 |
1011.45 |
85178.23 |
10032.59 |
|
100% concentration |
7995.49 |
9483.95 |
1258.61 |
109891.3 |
13054.34 |
|
120% concentration |
9885.17 |
11155.4 |
1476.71 |
138004.1 |
16010.38 |
|
Y=mx+C |
y = 393.85x + 107.5 |
y = 344.3x + 2576.5 |
y = 45.941x + 330.61 |
y = 5399.5x + 2993.3 |
y = 628.06x + 450.35 |
|
Corr.Coeff. |
0.9994 |
0.9998 |
0.9997 |
0.9994 |
0.9991 |
Precision:
Precision was evaluated in terms of intra-day repeatability and inter-day reproducibility. The intra-day repeatability was investigated using six separate sample solutions prepared, as reported above, from the freshly reconstructed tablet formulations at 100% of the target level. Each solution was injected in triplicate and the peak areas obtained were used to calculate means and RSD% values21. The % RSD for each solvent was found to be less than 10 and precision study was passed.
Figure 3: Calibration Graphs of Residual solvents present in Simeprevir API.
Figure-4: Typical Precision Chromatogram of Residual solvents present in Simeprevir API.
The accuracy (recovery) of Methanol, Toluene, Dichloromethane, Dichloroethane and Tetrahydrofuran (THF)in Simeprevir was conducted by spiking known quantities of each solvent into a sample, which is previously quantified for Methanol, Toluene, Dichloromethane, Dichloroethane and Tetrahydrofuran (THF) (during method precision stage) at various concentrations ranging from about 80 to 120% (80, 100, and 120%) of specification level. From accuracy data, the % recovery of residual solvents was found within the limits (90-110%) and % RSD for area did not exceed 10.0% for each solvent as per the ICH guideline. Results indicate that the method has an acceptable level of accuracy. The results are summarized in Table 5.
Figure-5(a) Accuracy Chromatogram (80% Level) of Residual solvents in Simeprevir API
Figure-5(b) Accuracy Chromatogram (100%Level) of Residual solvents in Simeprevir API.
Figure-5(c) Accuracy Chromatogram (120% Level) of Residual solvents in Simeprevir API.
Limits of detection (LOD) and quantification (LOQ) were estimated from the regression equation method. The detection limit was determined as the lowest concentration level resulting in a peak area of three times the baseline noise. 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 signal- to-noise 10.
Robustness:
To determine the robustness of the developed method, experimental conditions were deliberately changed and the relative standard deviations for replicate injections of all five residual solvents peaks were evaluated. The gaseous mobile phase flow rate was 2.5 mL/min. This was changed by ±0.5 units to 2.0 and 3.0 mL/min. The chromatographic variations were evaluated for theoretical plates and tailing factor for peaks of all five residual solvents were observed. From the above study upon changing the flow rates, theoretical plate count (NLT 2000) and tailing factor (NLT 2.0) were to be within limits.
Table-5: Recovery Study of working dilutions of Residual solvents present in Simeprevir API
|
Accuracy |
Recovery % of Methanol |
Recovery % of Toluene |
Recovery % of Dichloro-methane |
Recovery % of Dichloro- ethane |
Recovery % of Tetrahydro- furan (THF) |
|
At 80% Level |
100.55 |
100.47 |
100.75 |
100.86 |
103.27 |
|
At 100% Level |
100.47 |
100.22 |
103.45 |
100.34 |
103.64 |
|
At 120% Level |
100.75 |
103.27 |
100.78 |
101.82 |
101.18 |
Table-6: LOD and LOQ data for the four Residual solvents present in Simeprevir-API.
|
Parameter |
Methanol |
Toluene |
Dichloro- methane |
Dichloro-ethane |
Tetrahydro-furan (THF) |
|
LOD |
4μg/mL |
3μg/mL |
0.4μg/mL |
5μg/mL |
3.5μg/mL |
|
LOQ |
12.5μg/mL |
9.5μg/mL |
1.18μg/mL |
15μg/mL |
12μg/mL |
CONCLUSION:
In summary, a headspace gas chromatographic method was established and validated for the determination of five residual solvents in Simeprevir. The established method shows high sensitivity, good accuracy and linearity, which was applied successfully in the quality control of three batches of Simeprevir. A single, rapid and highly selective HSGC method was developed and validated for the quantification of residual solvents present in Simeprevir API through an understanding of the synthetic process, nature of solvents and nature of stationary phases of columns. The residual solvents Methanol, Tetrahydrofuran, Toluene, Dichloromethane and Dichloroethane were determined. The developed method is specific, accurate, precise and rugged as per ICH guidelines.
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Received on 25.09.2020 Modified on 17.10.2020
Accepted on 14.11.2020 © RJPT All right reserved
Research J. Pharm. and Tech. 2021; 14(10):5175-5181.
DOI: 10.52711/0974-360X.2021.00900