Dispersive liquid–liquid microextraction combined with gas chromatography for extraction and determination of class 1 residual solvents in pharmaceuticals

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<ul><li><p>J. Sep. Sci. 2012, 35, 10271035 1027</p><p>Mir Ali Farajzadeh1Leila Goushjuii1Djavanshir Djozan1Javad Kompani Mohammadi2</p><p>1Department of AnalyticalChemistry, Faculty of Chemistry,University of Tabriz, Tabriz, Iran</p><p>221st Street, Kaveh IndustrialCity, Mahban PharmaceuticalCompany, Tehran, Iran</p><p>Received October 25, 2011Revised January 10, 2012Accepted January 10, 2012</p><p>Research Article</p><p>Dispersive liquidliquid microextractioncombined with gas chromatography forextraction and determination of class 1residual solvents in pharmaceuticalsThe present study reports a new method for analyzing class 1 residual solvents (RSs), 1,1-dichloroethene (1,1-DCE), 1,2-dichloroethane (1,2-DCE), 1,1,1-trichloroethane (1,1,1-TCE),carbon tetrachloride (CT), and benzene (Bz), in pharmaceutical products using dispersiveliquidliquid microextraction (DLLME) combined with gas chromatographyflame ioniza-tion detection (GC-FID). Unlike common DLLME methods, solvents of high boiling pointwere selected as dispersive and extraction solvents in order to prevent their chromato-graphic peaks from overlapping with those of analytes that have short retention times.Therefore N,N-dimethyl formamide (DMF) and 1,2-dibromoethane (1,2-DBE) were chosenas dispersive and extraction solvents, respectively. Analytical parameters of the proposedmethod were determined and good linearities and broad linear ranges (LRs) were ob-tained. Taking 500 mg samples, limit of detections for the tested pharmaceuticals wereobtained as 0.11, 0.03, 0.05, 0.05, and 0.006 g g1 for CT, 1,1-DCE, 1,2-DCE, 1,1,1-TCE,and Bz, respectively, which are considerably much lower than their permissible limits inpharmaceuticals.</p><p>Keywords: Class 1 residual solvents / Dispersive liquidliquid microextraction /Gas chromatography / PharmaceuticalsDOI 10.1002/jssc.201100917</p><p>1 Introduction</p><p>Residual solvents (RSs) are defined as volatile organic com-pounds (VOCs) that are used in or produced during the man-ufacturing process of drug substances, or in the preparationof drug products. These solvents are not completely removedby practical manufacturing techniques such as freezedryingand drying at high temperature under vacuum. The presenceof these unwanted chemicals even in small amounts, notonly may influence the efficacy and safety of drug (regardingboth human health and environmental issues), but also theirchemical identity and amountmay affect some physicochem-ical properties of drug products such as: their particle size,</p><p>Correspondence: Dr. Mir Ali Farajzadeh, Department of AnalyticalChemistry, Faculty of Chemistry, University of Tabriz, Tabriz, IranE-mail: mafarajzadeh@yahoo.com and mafarajzadeh@tabrizu.ac.irFax: +98-411-3340191</p><p>Abbreviations: Bz, benzene; CT, carbon tetrachloride; 1,2-DBE, 1,2-dibromoethane; 1,1-DCE, 1,1-dichloroethene; 1,2-DCE, 1,2-dichloroethane; DLLME, dispersive liquidliquidmicroextraction; DMF, N,N-dimethyl formamide; EF, en-richment factor; ERs, extraction recoveries; GC-FID, gaschromatography-flame ionization detection; LR, linear range;RSs, residual solvents; 1,1,1-TCE, 1,1,1-trichloroethane;VOCs, volatile organic compounds</p><p>crystalline structure [1], wettability [2, 3], stability, and disso-lution properties [4]. Moreover, RSs may play a key role inthe modification of product odor as well [5]. This implies thatquality control of pharmaceutical products should include ob-taining accurate information on identity and quantity of anyRS present.</p><p>Guideline Q3C [6] was adopted by the International Con-ference on Harmonization (ICH) of Technical Requirementsfor Registration of Pharmaceuticals for Human Use on 17July 1997 and it has been accepted by the European Phar-macopoeia (5th Ed.), Japanese Pharmacopoeia (14th Ed.),the United States Pharmacopoeia (28th Ed.), and the Chi-nese Pharmacopoeia 2005. It classified RSs into three cate-gories according to their potential toxicity and limited theiramount in pharmaceuticals. Class 1 includes solvents con-sidered to be the most toxic, such that their use should beavoided in the production of pharmaceutical products. Thesechemicals are: benzene (Bz), carbon tetrachloride (CT), 1,2-dichloroethane (1,2-DCE), 1,1-dichloroethene (1,1-DCE), and1,1,1-trichloroethane (1,1,1-TCE) (the latter owing to its ad-verse environmental impact). However, if their use is un-avoidable, their level should be restricted to the amounts givenin Table 1 [6]. Class 2 and class 3 RSs are considered to be oflesser hazard. A list of other RSs that may attract a growinginterest could someday be called class 4 RSs.</p><p>As mentioned above, the RSs are VOCs; thereforethey can be separated and determined qualitatively and</p><p>C 2012 WILEY-VCH Verlag GmbH &amp; Co. KGaA, Weinheim www.jss-journal.com</p></li><li><p>1028 M. A. Farajzadeh et al. J. Sep. Sci. 2012, 35, 10271035</p><p>Table 1. Class 1 residual solvents (RSs), related applied symbolsin this article, ICH recommended concentration and im-pact of their toxicity</p><p>Compound ICH recommended Concernconcentration (ppm)</p><p>Carbon tetrachloride(CT)</p><p>4 Carcinogen</p><p>1,1,1-Trichloroethane(1,1,1-TCE)</p><p>1500 Toxic andenvironmental hazard</p><p>1,1-Dichloroethene(1,1-DCE)</p><p>8 Toxic</p><p>Benzene (Bz) 2 Toxic1,2-Dichloroethane</p><p>(1,2-DCE)5 Environmental hazard</p><p>quantitatively by gas chromatography (GC). So, direct injec-tion to GC system has commonly used for achieving thispurpose [7]. Furthermore, use of dynamic headspace sys-tem, i.e. purge and trap [3, 8], and programmed tempera-ture vaporizer inlet to inject the samples into the chromato-graphic column [911] are previously devised and appliedmethods. However it should be considered that complex ma-trix of pharmaceutical samples and low concentrations ofanalytes (their prescribed limits are at ppm levels), make itnecessary to introduce an isolation and/or preconcentrationstep in the analytical procedure. Solid-phase microextraction(SPME) coupled with GC has been established and appliedto determine RSs in drugs being soluble in water [1214].Headspace-solid phase microextraction (HS-SPME) [1517]has also been used in this field. A headspace-liquid phase mi-croextraction (HS-LPME) combinedwithGCwas proposed byWang et al. in 2006 for extraction anddetermination of volatilesolvent residues in pharmaceutical products [18]. Recently anew method was developed by combining the single-dropmicroextraction (SDME) and multiple headspace extractionfollowed by capillary GC-FID for quantitative determinationof volatile RSs in solid drug [19].</p><p>The above-mentioned methods, despite their applicabil-ity, have some defects. Direct injection method is simple butits main disadvantage is that non-volatile components arealso injected into the system which leads to injector con-tamination, column contamination, and deterioration withunavoidable matrix effects [16,20]. Headspace injection is analternative technique, but it is rather limited in terms of opti-mization possibilities with respect to its selectivity [16]. SPMEalso suffers from some disadvantages. That uses expensivematerials, is time-consuming, and usually has carryover ef-fects [21].</p><p>A few years ago, a new liquidliquid microextrac-tion method named dispersive liquidliquid microextraction(DLLME) was introduced by Assadi and co-workers [22] asan extraction and preconcentration method which has manybenefits and eliminates most disadvantages of the traditionalsample preparation techniques. Owing to the outstandingmerits of DLLME including simplicity, low cost, rapidity and</p><p>high enrichment factor (EF), this technique was widely ac-cepted and has been successfully applied in the preconcen-tration of different target compounds in aqueous samples[2228].</p><p>In the present work, we developed this strategy and com-bined itwithGC-FID for the extraction, preconcentration, anddetermination of toxic ICH class 1 solvents in pharmaceuticalproducts. By now, DLLME was not used in determination ofvolatile compounds such as solvents, and all reported worksare based on extraction and dispersive solvents of low boil-ing point. In this work, common dispersive solvents such asacetone, methanol, and acetonitrile and common extractionsolvents such as CT, chloroform, etc. cannot be used. Chro-matographic peaks of the target analytes (volatile solvents) areoverlapping with those of solvents used in DLLME. For thefirst time, dispersive and extraction solvents (e.g. DMF and1,2-DBE, respectively) of boiling point higher than that ofanalytes were used in DLLME that decrease or eliminate sep-aration problems in GC. The proposed method is rapid andsimple, uses mg levels of sample and L levels of extractionand dispersive solvents and has low LODs.</p><p>2 Experimental</p><p>2.1 Chemicals and samples</p><p>The solvents used were purchased from the followingsources: CT, 1,2-DCE, 1,2-DBE, 1,1,1-TCE, N,N-dimethylformamide (DMF), dimethyl sulfoxide (DMSO), methanol,acetone, acetonitrile, 1,1-DCE, and n-propanol were fromMerck (Darmstadt, Germany); and 1,2-bromochloroethane,1,1,2,2-tetrachloroethane, and 1,1,2,2-tetrabromoethane werefrom Janssen (Beerse, Belgium). Other chemicals such assodium chloride, hydrochloric acid, and sodium hydrox-ide were obtained from Merck. Deionized water (GhaziCompany, Tabriz, Iran) was used for preparation of aque-ous solutions. Erythromycin and clindamycin hydrochlo-ride were from Sepidaj Pharmaceutical Company (Tehran,Iran), cefepime was from Hetro (Hyderabad, India), amoxi-cillin trihydrate was from Farabi Company (Isfahan, Iran),ceftriaxone-Na was from Daana Pharmaceutical Company(Tabriz, Iran), and meglumine compound 76% (sodium di-atrizoate/meglumine diatrazoate, 10:66) was from DarouPakhsh Company (Tehran, Iran).</p><p>2.2 Solutions</p><p>To optimize the preconcentration and chromatographic sep-aration, standard solution of analytes (class 1 RSs) was pre-pared in 1,2-DBE ormethanol with concentrations as follows:1,1-DCE, 5000; 1,2-DCE, 5000; 1,1,1-TCE, 5000; CT, 25 000;and Bz, 1000 g mL1. Standard solution in 1,2-DBE wasdaily injected into the separation system (three times) andthe obtained peak areas were used in calculation of extrac-tion recoveries (ERs) and EFs. Stock solution of analytes in</p><p>C 2012 WILEY-VCH Verlag GmbH &amp; Co. KGaA, Weinheim www.jss-journal.com</p></li><li><p>J. Sep. Sci. 2012, 35, 10271035 Sample Preparation 1029</p><p>methanol was used for preparation of working aqueous solu-tions by appropriate dilutions. These solutions were preparedjust before analysis.</p><p>2.3 Sample preparation</p><p>One liquid sample and five solid pharmaceutical samples in-dicated in section 2.1 were analyzed. The liquid sample (meg-lumine compound) was diluted by two-folds with deionizedwater and 5 mL of it was used. The pH was 5.6. In the casesof solid samples, 500 mg of them were dissolved in 5-mLdeionized water (cefepime, ceftriaxone-Na, and clindamycinhydrochloride) or in 0.5 M NaOH (erythromycin and amoxi-cillin trihydrate) and were used as sample solutions. For eachsample, mean of three replicate analyses was taken as thedetermination result.</p><p>2.4 Instrumentation and chromatographic conditions</p><p>Chromatographic analysis was performed on a gas chromato-graph (GC-15A, Shimadzu, Kyoto, Japan) equipped with asplit/splitless injection system, and a flame ionization detec-tor. Helium (99.999 %, Gulf Cryo, United Arab Emirates)was used as the carrier gas at a constant linear velocity of30 cm s1. Injection was carried out in a split mode with asplit ratio of 1:10. Compounds were separated on a capillarycolumn SPTM-2380 (60 m 0.25 mm i.d. and 0.2 m filmthickness) (Supelco, Bellefonte, PA, USA). Oven temperaturewas programmed as follows: 30C for 8min, ramped increaseat a rate of 20Cmin1 up to 200C and the final temperatureheld for 4min. The total time for oneGC runwas 20min. Thetemperature of FID and injector were maintained at 200C.The injection volume was 0.5 L. Hydrogen gas generatedwith a hydrogen generator (OPGU-1500s, Shimadzu, Japan)was used for FID at a flow rate of 40 mL min1. Flow rateof air was 300 mL min1. Hettich centrifuge, model D-7200(Germany) was used for acceleration of phase separation.</p><p>2.5 Procedure</p><p>To perform DLLME, 5-mL aliquot of aqueous standard orsample solution was placed into a 10-mL glass tube with aconical bottom and the opening of the tube was then tightlyclosed with parafilm. Then, the needle of a 1-mL syringecontaining 100 L of DMF as disperser and 25 L of 1,2-DBEas extraction solvent, was penetrated through the parafilmand its content was rapidly injected into the aqueous solution.The mixture was centrifuged at a rate of 3000 rpm for 3 min.The centrifugation allowed the organic phase (10 1 L)to settle in the bottom of the conical test tube. An aliquot(0.5 L) of the organic phase was removed using a 1-L GCsyringe (zero dead volume, Hamilton, Switzerland) and wasinjected into GC system for analysis.</p><p>2.6 Calculation of EF, extraction recovery (ER), andrelative recovery (RR)</p><p>EF is expressed as the ratio of the analyte concentration in theextraction phase (Csed) to the initial concentration of analytein sample solution (C0). ER is defined as the percentage oftotal analyte amount extracted to the extraction phase and canbe calculated according to the following equation.</p><p>ER% = (nsed/n0) 100 = (CsedVsed/C0V0) 100= E F (Vsed/V0) 100 (1)</p><p>where n0 is the initial amount of analyte in the sample, nsedis the amount of analyte in the extraction phase, and V0 andVsed are volumes of the sample and the sedimented phase,respectively.</p><p>The RR can be calculated from the following equation:</p><p>RR% = [(Cfound Creal)/Cadded] 100 (2)where Cfound, Creal, and Cadded are the obtained concentra-tions of analyte after performing the proposed method onthe real sample into which a known amount of standard wasadded, the concentration of analyte in real sample, and theconcentration of standard that was spiked into the real sam-ple, respectively. Cadded is a predetermined amount and Cfoundand Creal are determined by taking advantage of a calibrationgraph [2931].</p><p>3 Results and discussion</p><p>3.1 Extraction solvent selection</p><p>For developing an efficient DLLME method, choosing an ap-propriate extraction solvent is of vital importance. Generallyspeaking, the extraction solvent used in DLLME proceduresmust fulfill the following requirements: it should have a den-sity higher or lower than water density, low solubility in wa-ter, high extraction capability for the target analytes, goodchromatographic behavior, and finally it should be easily dis-persed in water during dispersing step. Based on the aboverequirements, and considering this fact that the analytes havelow boiling points, some halogenated solvents of relativelyhigh boiling points, including 1,1,2,2-tetrabromoethane (b.p.244C), 1,2-bromochloroethane (b.p. 107C), 1,2-DBE (b.p.132C), and 1,1,2,2-tetrachloroethane (b.p. 146.5C), havingdifferent polarities were tested as possible extraction sol-vents. A series of experiments were carried out using 1.0-mLDMF (as dispersive solvent) mixed with different volumes ofthe extraction solvent. Our goal was to achieve sedimentedphase volume of 10 L when the mixture was rapidly in-jected into 5-mL...</p></li></ul>