Development and validation of a headspace gas chromatographic method for the determination of residual solvents in arterolane (RBx11160) maleate bulk drug

Abstract

Objective:

Arterolane maleate is an antimalarial drug currently under Phase III clinical evaluation, and presents a simple, economical and scalable synthesis, and does not suffer from safety problems. Arterolane maleate is more active than artemisinin; and is cheap to produce. It has a longer lifetime in the plasma, so it stays active longer in the body. To provide quality control over the manufacture of any API, it is essential to develop highly selective analytical methods. 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 Arterolane Maleate bulk drug.

Materials and Methods:

The method development and its validation were performed on Perkin Elmer's gas chromatographic system equipped with Flame Ionization detector and head space analyzer. The method involved a thermal gradient elution of ten residual solvents present in arterolane maleate salt in RTx-624, 30 m × 0.32 mm, 1.8 μ column using nitrogen gas as a carrier. The flow rate was 0.5 ml/min and flame ionization detector (FID) was used.

Results:

During method validation, parameters such as precision, linearity, accuracy, limit of quantification and detection and specificity were evaluated, which remained within acceptable limits.

Conclusions:

The method has been successfully applied for the quantification of the amount of residual solvents present in arterolane maleate bulk drug.The method presents a simple and reliable solution for the routine quantitative analysis of residual solvents in Arterolane maleate bulk drug.

The need for a rapid and reliable method for the determination of residual solvents has become significant due to the toxicity of residual solvents in drug substances and drug products.[1] 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 industry. 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 solvents.[2] Many pharmaceutical products have to be analyzed for residual solvents at different stages of their development (raw materials, intermediate products and final product). Organic solvents such as ethanol, dichloromethane, hexanes, n-heptane, etc. are frequently used in the pharmaceutical industry. The manufacturing of new active pharmaceutical ingredients (APIs) under Good manufacturing practices (GMP) conditions demands adequate control of quality of the different ingredients used in the synthesis. Organic residual solvents have therefore to be controlled during any GMP synthesis. Headspace gas chromatography (HSGC) is the most favored technique for the analysis of volatiles and semivolatile organics in solid, liquid and gas samples.[3–7]

Arterolane maleate [Figure 1] is an antimalarial drug currently under Phase III clinical evaluation, and presents a simple, economical and scalable synthesis, and does not suffer from safety problems.[8] Arterolane maleate contains a type of bridge that is considered important for its activity (it generates radicals that target proteins in the parasite), and was engineered with the bulky adamantane group next to it to protect the peroxide bridge.[9] Arterolane maleate is more active than artemisinin; it has sufficient solubility to be administered orally, and is cheap to produce. It has a longer lifetime in the plasma, so it stays active longer in the body.[10–12] To provide quality control over the manufacture of any API, it is essential to develop highly selective analytical methods. HSGC is one of the most widely accepted techniques used to monitor the residual solvents present in APIs. Therefore, a highly selective, rapid and linear HSGC method was developed for the quantification of a total of 10 residual solvents which are used during the synthesis and manufacturing of arterolane maleate in accordance with International Conference on Harmonization (ICH) guidelines.[13,14]

Figure 1.

Structure of arterolane maleate

Materials and Methods

Chemical and reagents

Arterolane maleate was obtained from NCE Scaleup division of Ranbaxy Research Laboratories, Gurgaon, India. Milli Q water was obtained from inhouse Milli Q water plant. N,N-dimethyl formamide, sodium chloride, pentane, ethanol, 2-methylpentane, dichloromethane, 3-methylpentane, n-hexane, methylcyclopentane, cyclohexane, benzene and n-heptane were purchased from Fluka Chemical Co., Inc. (Milwaukee, WI, USA).

Apparatus and chromatographic conditions

The analysis for the determination of residual solvents and its validation was performed on Perkin Elmer's gas chromatograph Clarus 500 equipped with turbomatrix headspace sampler and a flame ionization detector (FID).

RTx-624 with dimensions of 30.0 m × 0.32 mm using a 1.8 μm capillary column, from Restek Corporations was used for the validation. The final validated HSGC method for the separation of residual solvents used a flow rate of 0.5 ml/min. Oven temperature was maintained at 40°C for 20 min, and then a linear thermal gradient of 15°C/min to 200°C was used with a final hold of 5 min. Total run time was 35.0 min. Nitrogen was used as a carrier gas at a constant flow rate of 0.5 ml/min. The headspace and detector conditions used for the analysis are outlined in Tables 1 and 2, respectively.

Table 1.

Headspace conditions

Table 2.

Detector conditions (FID)

Preparation of standard and sample vials

A common standard stock solution in N,N-dimethylformamide containing all the known residual solvents of arterolane maleate API (i.e., pentane, ethanol, 2-methylpentane, dichloromethane, 3-methylpentane, n-hexane, methylcyclopentane, cyclohexane, benzene and n-heptane) was prepared in such a way that it has a final concentration of 50 μg/g for pentane, 2-methylpentane, 3-methylpentane, n-hexane, cyclohexane, methylcyclopentane; 3000 μg/g for ethanol, 375μg/g for dichloromethane, 1500 μg/g for n-heptane and 2 μg/g for benzene. The standard vial was prepared with 0.2 ml of the standard solution and the sample vials were prepared with approximately 0.1 g of sample with 0.2 ml N,N-dimethylformamide as diluent. Water and sodium chloride were added in both the sample and standard vials in a ratio of 2 : 1, respectively.

Method validation

The method validation was done by evaluating specificity, limit of detection (LOD) and limit of quantitation (LOQ), linearity, accuracy, repeatability, and method precision of residual solvents as indicated in the ICH guideline Q2B ‘Validation of Analytical Procedures: Methodology.’[15]

Specificity

Arterolane API sample was spiked with pentane, ethanol, 2-methylpentane, dichloromethane, 3-methylpentane, n-hexane, methylcyclopentane, cyclohexane, benzene and n-heptane individually and each sample was chromatographed to examine interference, if any, of the residual solvents’ peak with each other.

Linearity

The linearity of the method was determined by making injections of each residual solvent over the range of limit of quantification (LOQ) to 200% of the standard concentration as mentioned earlier.

System precision

Six vials of standard solution were prepared and chromatographed in the GC system.

Method precision

A single batch of arterolane (RBx11160) API was prepared six times and analyzed by the proposed method.

Detection and quantification limits

The LODs of residual solvents in arterolane (RBx11160) maleate were determined based on signal-to-noise ratio of 3 : 1. The LOQs of residual solvents were determined based on signal-to-noise ratio 10 : 1. Six replicates were performed at each level.

Accuracy

Known amount of sample (about 100 mg) was taken separately in nine different vials and spiked with known quantities of pentane, ethanol, 2-methylpentane, dichloromethane, 3-methylpentane, n-hexane, methylcyclopentane, cyclohexane, benzene and n-heptane at three different levels, in triplicate.

System suitability

A system suitability criterion was taken to be the resolution between the critical pairs, i.e., 2-methylpentane and dichloromethane. The system suitability was evaluated by injecting the standard solution on various days before starting the any exercise during validation study.

Results and Discussion

Method development

An understanding of the nature of the various residual solvents present in API is the foremost prerequisite for successful method development in HSGC. In addition, successful method development should result in a fast, simple and time efficient method that is capable of being utilized in a manufacturing setting. Following were the stepwise strategies for the method development in our case.

Column selection

The primary goal of column selection was to resolve a total of 10 residual solvents (i.e., pentane, ethanol, 2-methylpentane, dichloromethane, 3-methylpentane, n-hexane, methylcyclopentane, cyclohexane, benzene and n-heptane) which were used during the synthesis and manufacturing of arterolane maleate. Several columns were initially investigated to finalize a single method for the separation and quantitation of solvents. Wall-coated capillary columns of various brands with a variety of phases and dimensions have been investigated, e.g., column A (30 m length, 0.32 mm i.d. with a stationary phase of 6% cynopropyl phenyl and 94% dimethyl polysiloxane film of 3.0 μ), RTx-624 (30 m length, 0.32 mm i.d. with a stationary phase of 6% cynopropyl phenyl and 94% dimethyl polysiloxane film of 1.8 μ). In the above column, the response was found to be comparatively lower and peak shapes were found to be unsatisfactory. Also, there was a problem in resolving the closely related peaks specially 2-methylpentane and dichloromethane. Therefore, RTx-624 with dimensions of 30 m × 0.32 mm, 1.8 μ proved to be the best column that could fulfill all the needs of the method, i.e., higher sensitivity, shorter runtime and higher resolution between the critical pairs.

Thermal program and thermal gradient

A linear thermal gradient was chosen to provide elution of the solvents’ peak during the isothermal segment of the chromatographic run for better quantification. An initial hold of 20 min at 40°C and a linear thermal gradient to 200°C at 15°C/min was found to give the best peak shape and retention without affecting the resolution. The critical pair under these conditions was 2-methyl pentane and dichloromethane for which resolution was achieved more than 2.

Headspace method optimization

The headspace method was optimized in such a way that maximum amount of the solvents present in the sample get evaporated for the detection. For this the standard and sample vials were heated at 60–95°C for 20–30 min with constant shaking. A combination of sample vial heating at 75°C with 20 min shaking was found to be suitable for getting a good response. Table 1 shows the complete headspace condition.

Method validation

Specificity

The relative retention time of the all-residual solvents indicated that they were well separated from each other [Table 3]. A typical chromatogram of standard solution is shown in Figure 2.

Table 3.

Specificity

Figure 2.

Chromatogram of residual solvents of arterolane maleate

Linearity

The FID detector response was shown to be linear over this range with correlation coefficients (R²) higher than 0.99 [Table 4].

Table 4.

Linearity of all the residual solvents over the range of LOQ to 200%

System precision

Results indicate an acceptable level of precision for the analytical system [Table 5]. The % relative standard deviation (%RSD) of area obtained from six standard vials was calculated to show the system precision. An acceptance criterion for system precision was taken to be not more than (NMT) 8% for six vials.

Table 5.

System precision

Method precision

The %RSD values indicate that the method has an acceptable level of precision. The %RSD values of area obtained from six sample vials were calculated to show the method precision [Table 6]. An acceptance criterion for method precision was taken to be NMT 10% for six preparations.

Table 6.

Method precision

Detection and quantification limits

Tables 7 and 8 show the quantification and detection limits, respectively, for the samples.

Table 7.

Limit of quantification

Table 8.

Limit of detection (LOD)

Accuracy

The accuracy was evaluated by the % recoveries of residual solvents spiked in the sample. An acceptance criterion for accuracy was that the recovery should be in the range of 80-120%. The recoveries of residual solvents ranged between 93.06 and 106.6%. Results indicate that the method has an acceptable level of accuracy [Table 9].

Table 9.

Accuracy

System suitability

Using the system suitability software, resolution between 2-methylpentane and dichloromethane was calculated. The criterion for system suitability was that the resolution between critical pair mentioned above should not be less than 1.5 and it was found well above the minimum passing limit [Table 10].

Table 10.

System suitability

Conclusion

A single, rapid and highly selective HSGC method was developed and validated for the quantification of residual solvents present in arterolane maleate bulk drug through an understanding of the synthetic process, nature of solvents and nature of stationary phases of columns. The residual solvents pentane, ethanol, 2-methylpentane, n-heptane, 3-methylpentane, n-hexane, methylcyclopentane, cyclohexane, benzene and dichloromethane were determined. The method was shown to be specific for arterolane maleate and was applied successfully to monitor and control these solvents on a manufacturing level. The method was found to be applicable for the routine analysis of the arterolane maleate in pharmaceutical industry.