Chromatography 2015, 36, Technical Note

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1 Technical Note Development and Validation of an Analytical Method for Simultaneous Quantitation of Organic Volatile Impurities in Technical-Grade Active Ingredients of Pesticides and Agrochemicals by Headspace Gas Chromatography Masato KAZUSAKI *, Satoshi OKUMURA, Kozo KITA, Makiko MUKUMOTO, Masahiko OKAMOTO Analytical Science Group, Organic Synthesis Research Laboratory, Sumitomo Chemical Co., Ltd., 1-98, Kasugade-naka 3-chome, Konohana-ku, Osaka , Japan Abstract A simple, rapid, accurate, precise headspace gas chromatographic (HS-GC) method was developed and validated for simultaneous quantitation of organic volatile impurities (residual solvents) in technical-grade active ingredients (TGAIs) of pesticides and agrochemicals. The HS-GC method utilizes conventional capillary columns. The HS-GC operating conditions were optimized, and 18 organic volatile impurities were efficiently separated. The versatility of the method was confirmed in terms of validation characteristics of specificity, limit of quantitation, linearity, accuracy, and precision. The proposed method was successfully applied to the analysis of pesticide and agrochemical TGAIs. Keywords: Headspace gas chromatography; Validation; Residual solvent; Pesticide; Agrochemical 1. Introduction Organic solvents are routinely used in the manufacture of technical-grade active ingredients (TGAIs) for pesticides and agrochemicals. To remove organic solvents after isolation of the TGAIs, a drying step is usually involved in the manufacturing process. However, even after the drying step, traceable amounts of organic solvents remain in the TGAIs. These residual solvents are undesirable owing to the toxicity, and negatively impact the quality of the TGAIs. To promote reduction of the potential risk posed to human health and the environment, the United States Environmental Protection Agency (EPA) has classified organic volatile impurities into four toxic categories, Class I Class IV, based on their toxicity [1]. The International Conference on Harmonization of technical requirements for registration of pharmaceuticals for human use (ICH) has also set forth a guideline, Impurities: Guideline for Residual Solvents (Q3C) [2] for controlling residual solvents, and proposed permitted daily exposure (PDE) levels for residual solvents contaminated in drug substances and drug products. The PDE levels were set on the basis of toxicity studies. For -67- quality control, manufacturers of drug substances, pesticides, and agrochemicals routinely establish testing methods to quantitate the content of organic volatile impurities in TGAIs. However, the quantitation of organic volatile impurities in TGAIs is known to be one of the most tedious analytical tasks in the industry. Headspace gas chromatography (HS-GC) is the most favorable technique for the analysis of organic volatile impurities in solid, liquid, and gas samples. The volatile components in the liquid or solid sample are transferred to a closed vial, and kept to reach equilibrium between the sample and the vapor in the headspace. A fraction of the headspace vapor is sampled and introduced into a GC system. Injection of the analytes evaporated from the sample into the GC system could minimize the contamination of the GC instrument and deterioration of the GC capillary column [3-9]. A comprehensive analytical procedure using HS-GC for residual solvents in drug substances and drug products is described in the United States Pharmacopeia 37 (USP 37) [10]. The analytical procedures in the USP HS-GC method are divided into two sections, for water-soluble and * Corresponding author: Masato KAZUSAKI Received: 30 May 2015 Tel: ; Fax: Accepted: 22 June kazusakim@sc.sumitomo-chem.co.jp J-STAGE Advance Published: 6 July 2015 DOI: /jpchrom

2 water-insoluble substances. The USP approach is typically a limit test for residual solvents that are listed in ICH Q3C, and implementation of this method is a subject of debate, due to the limited specificity. Comprehensive analytical procedures for organic volatile impurities present in pesticides and agrochemicals have not been reported, to the best of our knowledge. The purpose of this study was to establish a new, exhaustive analytical procedure based on HS-GC to quantitate the organic volatile impurities occurring in these TGAIs in a single injection. This report describes the development, validation, and application of a simultaneous analytical method for quantitation of organic volatile impurities in TGAIs for pesticides and agrochemicals. 2. Experimental 2.1. Materials and reagents Methanol of HPLC grade was purchased from Nacalai Tesque, Inc. (Kyoto, Japan). Analytical-grade ethanol, acetone, hexane, ethylacetate, 1-butanol, toluene, ethylbenzene, o-xylene, m-xylene, p-xylene, and HPLC-grade 2-propanol, acetonitrile, and tetrahydrofuran were obtained from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Analytical-grade tert-butylmethylether, heptane, and dimethylformamide were from Kanto Chemical Co, Inc. (Tokyo, Japan). 1-Propanol, 2-butanol, methylisobutylketone, isobutylacetate, and butylacetate of analytical grade were purchased from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). TGAIs of pesticides and agrochemicals were from Sumitomo Chemical Co., Ltd. (Tokyo, Japan) Instruments A gas chromatograph (GC-2010 Plus; Shimadzu Scientific Instruments, Kyoto, Japan) equipped with a flame ionization detector was used for analysis. A headspace autosampler HT2000H (ALPHA M.O.S. Japan K.K., Tokyo, Japan) was used to load the analytes. Capillary columns DB-WAX (0.5 µm thickness, 60 m length, 0.25 mm i.d.) and DB-5 (1 µm thickness, 30 m length, 0.25 mm i.d.) from Agilent Technologies Japan, Ltd. (Tokyo, Japan), and OVI-G43 (3.0 µm thickness, 30 m length, 0.53 mm i.d.) from Sigma-Aldrich Japan (Tokyo, Japan) were employed in this study Headspace gas chromatographic conditions Vials were heated at 100ºC for 10 min with shaking. A 1-mL fraction of the vapor in the headspace of the vial was injected into the GC injection port. The temperature of the injection port was maintained at 225ºC at a split ratio of 1:5, with helium gas as a carrier. The flow rate was maintained at 5.0 ml/min. The detector temperature was set at 270ºC. The column temperature was maintained at 40ºC for three minutes, and raised at a rate of 10ºC min -1 to 65ºC, held for 3 minutes, then increased at a rate of 5ºC min -1 to 85ºC, held for 2 minutes, finally increased at a rate of 30ºC min -1 up to 220ºC Validation of analytical procedure The validation study was carried out by evaluating the validation characteristics of specificity, linearity, limit of quantitation (LOQ), accuracy, and precision (repeatability), based on the ICH guideline Q2 [11] and the Health and safety executive guideline [12] focusing on validation of analytical procedures. These guidelines indicate the concentration range to be examined in the individual validation studies (linearity, accuracy and precision). Based on the concept of these guidelines, the range for the validation study for the analytical procedure on quantitation of organic volatile impurities should be defined based on the PDE level stipulated in ICH guideline Q3C. Dimethylformamide was selected as the diluent for preparation of the standard and sample solutions, because of its ability to dissolve a wide range of chemicals. In this study, the sample solutions were prepared by dissolving 100 mg of the TGAI in 1 ml of dimethylformamide. Stock solutions containing high levels of the organic volatile impurities (residual solvents) were prepared. To prepare the standard solutions for linearity study, the stock solutions were adequately diluted to prepare solutions containing the analyte in the range of 10% to 150% of the PDE level. For the accuracy and precision study, the stock solutions were diluted and spiked into the TGAIs at the concentration level of 10%, 100%, and 150% corresponding to the PDE Analyses of TGAIs for pesticides and agrochemicals Standard vials were prepared with 1 ml of each standard solution containing the PDE level of the respective residual solvents. Sample vials were also prepared with approximately 100 mg of each TGAI as the test samples and 1 ml of dimethylformamide as the diluent. 3. Results and discussion 3.1. Method development An analytical procedure was developed to quantitate the organic volatile impurities in TGAIs. Method development was carried out in a stepwise fashion, as follows: First, target organic solvents that would be employed in the manufacture of TGAIs were selected. Selection criteria were based on the viewpoint of toxicity of organic solvents and the expense for the manufacturer of the TGAI. In some cases, organic solvents employed in the manufacture of TGAIs for pesticides or agrochemicals are recovered in the manufacturing process, and used in the next manufacturing campaign of the TGAI. With the consideration of toxicity and expense [13], 18 organic solvents were listed as -68-

3 Peak area (µv.sec) Retention time (min) Fig. 1. Typical chromatogram showing the elution of organic solvents. 1, Methanol; 2, ethanol; 3, acetone; 4, 2-propanol; 5, acetonitrile; 6, tert-butylmethylether; 7, hexane; 8, 1-propanol; 9, ethylacetate; 10, 2-butanol; 11, tetrahydrofuran; 12, heptane; 13, 1-butanol; 14, methylisobutylketone; 15, toluene; 16, isobutylacetate; 17, butylacetate; 18, dimethylformamide, 19, ethylbenzene; 20, m-xylene, p-xylene; 21, o-xylene. Dimethylformamide (18) was employed as the diluent in this analytical procedure. Xylene that is employed in the TGAI manufacturing is a mixture of ethylbenzene, o-xylene, m-xylene and p-xylene. Under this operating procedure, ethylbenzene (19) and o-xylene (21) were eluted separately, but m-xylene and p-xylene (20) were co-eluted as the same peak. For the GC operating conditions, see the Experimental section. commonly employed solvents in TGAI manufacturing. Commonly, xylene for the TGAI manufacturing is a mixture of m-xylene (60%), p-xylene (14%), o-xylene (9%), and ethylbenzene (17%). In the next step, three types of columns, DB-WAX, DB-5, and OVI-G43, were compared for the analysis of organic volatile impurities by injecting all of the kinds of analytes investigated in this study. The column OVI-G43 selected in this investigation was coated with a thick film. By the developed HS-GC method, 18 organic solvents that are employed in the synthesis of TGAIs were separated in 20 minutes. Figure 1 shows a chromatogram demonstrating the separation of organic solvents investigated Method validation The validation process of the proposed method was carried out prior to quantitation of the organic volatile impurities in the TGAI, as follows: specificity, LOQ, and linearity were evaluated with 18 organic solvents. After evaluation of the specificity, LOQ, and linearity, recovery testing was performed above the LOQ level to evaluate the accuracy and precision for the proposed method with the actual TGAI for pesticides spiked with 18 organic solvents. In the next step, additional recovery testing was conducted with another real TGAI for agrochemicals spiked with some organic solvents used in the manufacturing of the TGAI, as a partial validation study (accuracy and precision) to estimate whether the proposed method is capable of application to that TGAI Specificity The retention time and resolution between the adjacent peaks in Fig. 1 are listed in Table 1. All the resolutions were more than 1.2. Ethylbenzene, m-xylene, p-xylene, and o-xylene were eluted after the dimethylformamide peak, and not completely resolved. So the amount of ethylbenzene and o-xylene should be calculated separately, and amounts of m-xylene and p-xylene be calculated together. Finally, the amount of xylene should be evaluated as the sum of individual amount of each component Linearity The linearity was investigated by making injections of each organic volatile impurity at five concentration levels ranging from 10% - 150% of the PDE level listed in Table 1. Calibration curves were constructed by plotting response -69-

4 Table 1. Specificity of organic volatile impurities. Organic volatile impurity ICH category Permitted daily exposure (ppm) Retention time (min) Resolution 1) Methanol Class ND 2) Ethanol Class Acetone Class Propanol Class Acetonitrile Class tert-butylmethylether Class Hexane Class Propanol Class Ethylacetate Class Butanol Class Tetrahydrofurane Class Heptane Class Butanol Class Methylisobutylketone Class Toluene Class Isobutylacetate Class Butyl acetate Class Dimethylformamide 3) 3) Ethylbenzene 4) Class m-xylene 4) Class 2 p-xylene 4) Class ) 1.9 o-xylene 4) Class ) Resolution was calculated with respect to the former eluting peaks. 2) Resolution was not determined because of the absence of the former peak. 3) Dimethylformamide was employed as the diluent. 4) Xylene for manufacturing usage is a mixture of m-xylene (60%), p-xylene (14%), o-xylene (9%), and ethylbenzene (17%). 5) m-xylene and p-xylene were not separated under this operating procedure. against concentration of organic volatile impurities, and statistical values of correlation coefficient, y-intercept, and were calculated, as well as the sum of residual squares, by using regression analysis. The rounded correlation coefficient values for the organic volatile impurities investigated were not less than 0.99, suggesting a sufficient linearity for the quantitation of amount of organic volatile impurities. The y-axis intercept and two-sided 95% were also calculated, as shown in Table 2. The of the y-axis intercept contained zero. All calibration curves were found to be essentially linear in the range examined, and passed through the origin Limit of quantitation The LOQ was determined by comparing the noise levels and the peak height of the analytes obtained from the standard solution for linearity study. The LOQ is the minimum concentration level at which a signal-to-noise ratio is not less than 10:1. Based on the signal-to-noise ratios of the peak height from the standard solution for linearity study and that of blank signals in the same chromatogram, the LOQ was established as the concentration level of 10% of the PDE level. Table 2. Linearity of organic solvents. y-intercept Organic volatile impurity Correlation coefficient Slope Lower limit Upper limit Methanol Ethanol Acetone Propanol Acetonitrile tert-butylmethylether Hexane Propanol Ethylacetate Butanol Tetrahydrofurane Heptane Butanol Methylisobutylketone Toluene Isobutylacetate Butylacetate Xylene 1) ) Statistical parameters were calculated as the sum of ethyl benzene, o-xylene, m-xylene, and p-xylene Accuracy and precision (repeatability) Accuracy and precision (repeatability) were assessed by analysis of the spiked samples and statistical of the recovery rates. A certain amount of a TGAI for pesticides was separately taken in 9 different vials, and dissolved in dimethylformamide containing analytes (organic volatile impurities) at three different levels, 10%, 100%, and 150% of the PDE level, in triplicate. Table 3 shows the average value (accuracy) and standard deviation (precision) of the recovery rates. The of the average recovery rate was calculated by using the Student s t-value, the average value, and the standard error of the mean. Precision was primarily estimated by applying ANOVA. of repeatability was obtained as the square root of square mean of residual error. The confidence interval of the standard deviation was also calculated, based on chi-square value, degrees of freedom, and sum of squares. As shown in Table 3, most of the 100% values of recovery rates are found within the two-sided 95% confidence intervals. The lower limits of the two-sided 95% confidence intervals for hexane and 1-butanol are 101.8% and 100.0%, respectively. These values are close to 100% recovery rates. These results indicate that only a negligible systematic error was determined in this analytical procedure. The standard deviations for the analytes were smaller than 20%, indicating this analytical procedure is sufficiently precise for use as a quantitation method of organic volatile impurities contained in a TGAI. As for accuracy and precision, the analytical procedure was shown to be conclusively acceptable. -70-

5 Table 3. Recovery of organic volatile impurities from the pesticide TGAI (%). Organic volatile impurity Recovery rate (accuracy) Standard deviation (repeatability) Two-sided 90% confidence interval Methanol < µ 1) < <σ 2) < 9.1 Ethanol < µ < <σ< 11.2 Acetone < µ < <σ< Propanol < µ < <σ< 11.7 Acetonitrile < µ < <σ< 6.7 tert-butylmethylether < µ < <σ< 3.3 Hexane < µ < <σ< Propanol < µ < <σ< 18.0 Ethylacetate < µ < <σ< Butanol < µ < <σ< 12.8 Tetrahydrofurane < µ < <σ< 4.8 Heptane < µ < <σ< Butanol < µ < <σ< 29.6 Methylisobutylketone < µ < <σ< 12.6 Toluene < µ < <σ< 9.7 Isobutylacetate < µ < <σ< 11.4 Butylacetate < µ < <σ< 14.3 Xylene 3) < µ < <σ< ) µ represents the population mean. 2) σis the population standard deviation. 3) Statistical parameters were calculated as the sum of ethyl benzene, o-xylene, m-xylene, and p-xylene Additional validation study with another TGAI The analytical procedure developed in this study was also applied to the quantitation of organic volatile impurities in another kind of TGAI for agrochemicals. To evaluate the interference arising from the TGAI, a partial validation study focusing on accuracy and repeatability was additionally performed, using six replicate analyses of the sample individually spiked with 100% level of organic volatile impurities that were utilized during the manufacture of the corresponding TGAI, according to ICH Q2. The results are listed in Table 4. Sufficient accuracy and precision levels were found in this study Quantitation of organic volatile impurities in actual TGAIs for pesticides and agrochemicals The analytical procedure was applied to batches of a TGAI for pesticides and agrochemicals. The contamination levels of organic volatile impurities in these TGAIs were lower than the PDE levels stipulated in ICH Q3C. 4. Conclusion A single, rapid, and highly selective HS-GC method was developed, fully validated, and applied to quantitation of residual organic volatile impurities in a TGAI for pesticides. For a TGAI for agrochemicals, the proposed HS-GC method was partially validated, and successfully applied to an actual TGAI. This analytical procedure could be applied to any TGAI after partial validation. Table 4. Recovery of organic volatile impurities calculated from six replicate analyses with the agrochemical TGAI. Recovery rate Standard deviation Organic (accuracy) (repeatability) volatile impurity Two-sided 90% Methanol < µ 1) < < σ 2) < 11.0 Heptane < µ < < σ < 4.7 Toluene < µ < < σ < 13.0 Xylene 3) < µ < < σ < ) µ represents the population mean. 2) σis the population standard deviation. 3) Statistic parameters were calculated as the sum of ethyl benzene, o-xylene, m-xylene, and p-xylene. References [1] United States Environmental protection agency: Inert Ingredients in pesticide Products Policy Statement, U.S. 52 Federal Register (FR) Notice 13305, [2] International Conference on Harmonization of Technical Requirements for registration of pharmaceuticals for Human use, Impurities: Guideline for residual Solvents, Q3C(R5) [3] Lindong, C.; Hua H.; Jing J.; Li F.; Fengmin L.; Qiliang, Q. J. Anal. Meth. Chem. 2013, 2013, 1-5. [4] Pandey, S.; Pandey, P.; Kumar, R.; Singh, N.P. Braz. J. Pharm. Sci. 2011, 47, [5] Michulec, M.; Wardencki, W. J. Chromatogr. A 2005, 1071, [6] Gupta, A.; Singh, Y.; Srinvias, K.; Jain, S.G.; Sreekumar, V. B.; Semwal, V. P. J. Pharm. Bioallied. Sci. 2010, 2, [7] Klick, S.; Sköld, A. J. Pharm. Biomed. Anal. 2004, 36, [8] Snow, N. H. Trends Anal. Chem. 2002, 21, [9] Camarasu, C.; Madichie, R. W. Trends Anal. Chem. 2006, 25, [10] United States Pharmacopoeia 37 <467>, Organic Volatile Impurities, [11] International Conference on Harmonization of Technical Requirements for registration of pharmaceuticals for Human use, Impurities: Guideline for residual Solvents, Validation of analytical Procedures: Text and Methodology, Q2(R1) [12] Health and safety executive: Guidelines for validation of analytical methods for non-agricultural pesticide active ingredients and products. [13] Kagaku Shohin (Chemical Products Handbook), The Chemical Daily: Tokyo, 2014; Chapters 9, 10, 24 and