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

J. Sep. Sci. 2012, 35, 1027–1035 1027

Mir Ali Farajzadeh1

Leila Goushjuii1Djavanshir Djozan1

Javad Kompani Mohammadi2

1Department of AnalyticalChemistry, Faculty of Chemistry,University of Tabriz, Tabriz, Iran

221st Street, Kaveh IndustrialCity, Mahban PharmaceuticalCompany, Tehran, Iran

Received October 25, 2011Revised January 10, 2012Accepted January 10, 2012

Research Article

Dispersive liquid–liquid microextractioncombined with gas chromatography forextraction and determination of class 1residual solvents in pharmaceuticals

The 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 dispersiveliquid–liquid microextraction (DLLME) combined with gas chromatography–flame 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 g−1 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.

Keywords: Class 1 residual solvents / Dispersive liquid–liquid microextraction /Gas chromatography / PharmaceuticalsDOI 10.1002/jssc.201100917

1 Introduction

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 freeze–dryingand 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 amount may affect some physicochem-ical properties of drug products such as: their particle size,

Correspondence: Dr. Mir Ali Farajzadeh, Department of AnalyticalChemistry, Faculty of Chemistry, University of Tabriz, Tabriz, IranE-mail: [email protected] and [email protected]: +98-411-3340191

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 liquid–liquidmicroextraction; 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

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.

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.

As mentioned above, the RSs are VOCs; thereforethey can be separated and determined qualitatively and

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1028 M. A. Farajzadeh et al. J. Sep. Sci. 2012, 35, 1027–1035

Table 1. Class 1 residual solvents (RSs), related applied symbolsin this article, ICH recommended concentration and im-pact of their toxicity

Compound ICH recommended Concernconcentration (ppm)

Carbon tetrachloride(CT)

4 Carcinogen

1,1,1-Trichloroethane(1,1,1-TCE)

1500 Toxic andenvironmental hazard

1,1-Dichloroethene(1,1-DCE)

8 Toxic

Benzene (Bz) 2 Toxic1,2-Dichloroethane

(1,2-DCE)5 Environmental hazard

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 [9–11] 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 [12–14].Headspace-solid phase microextraction (HS-SPME) [15–17]has also been used in this field. A headspace-liquid phase mi-croextraction (HS-LPME) combined with GC was proposed byWang et al. in 2006 for extraction and determination 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].

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].

A few years ago, a new liquid–liquid microextrac-tion method named dispersive liquid–liquid 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

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[22–28].

In the present work, we developed this strategy and com-bined it with GC-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.

2 Experimental

2.1 Chemicals and samples

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).

2.2 Solutions

To optimize the preconcentration and chromatographic sep-aration, standard solution of analytes (class 1 RSs) was pre-pared in 1,2-DBE or methanol with concentrations as follows:1,1-DCE, 5000; 1,2-DCE, 5000; 1,1,1-TCE, 5000; CT, 25 000;and Bz, 1000 �g mL−1. 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


J. Sep. Sci. 2012, 35, 1027–1035 Sample Preparation 1029

methanol was used for preparation of working aqueous solu-tions by appropriate dilutions. These solutions were preparedjust before analysis.

2.3 Sample preparation

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.

2.4 Instrumentation and chromatographic conditions

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 s−1. 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: 30�C for 8 min, ramped increaseat a rate of 20�C min−1 up to 200�C and the final temperatureheld for 4 min. The total time for one GC run was 20 min. Thetemperature of FID and injector were maintained at 200�C.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 min−1. Flow rateof air was 300 mL min−1. Hettich centrifuge, model D-7200(Germany) was used for acceleration of phase separation.

2.5 Procedure

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.

2.6 Calculation of EF, extraction recovery (ER), and

relative recovery (RR)

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.

ER% = (nsed/n0) × 100 = (CsedVsed/C0V0) × 100

= E F × (Vsed/V0) × 100 (1)

where n0 is the initial amount of analyte in the sample, nsed

is the amount of analyte in the extraction phase, and V0 andVsed are volumes of the sample and the sedimented phase,respectively.

The RR can be calculated from the following equation:

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 Cfound

and Creal are determined by taking advantage of a calibrationgraph [29–31].

3 Results and discussion

3.1 Extraction solvent selection

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.244�C), 1,2-bromochloroethane (b.p. 107�C), 1,2-DBE (b.p.132�C), and 1,1,2,2-tetrachloroethane (b.p. 146.5�C), 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 deionized water and then was centrifuged.Considering this goal, the results obtained showed that 27, 27,18, and 55 �L of 1,2-DBE, 1,1,2,2-tetrachloroethane, 1,1,2,2-tetrabromoethane, and 1,2-bromochloroethane were the ap-propriate volume of the extraction solvent, respectively, and



Figure 1. Effect of volumes of extrac-tion and dispersive solvents on the ex-traction efficiency. Conditions: sample:5-mL aqueous solution of 1,1-DCE (10�g mL−1), 1,2-DCE (10 �g mL−1), 1,1,1-TCE (10 �g mL−1), CT (50 �g mL−1), andBz (2 �g mL−1); centrifuging rate: 3000rpm, and centrifuging time: 3 min.

DLLME procedure was performed by the mentioned volumes.Experiments revealed that 1,2-DBE results in the highest an-alytical signals and a cleaner chromatogram in comparisonwith others. For this reason, 1,2-DBE was selected as the ex-traction solvent for further studies.

3.2 Selection of dispersive solvent

In DLLME, unlike other LPME techniques, the extraction ef-ficiency depends not only on the extraction solvent, but alsoon the type and volume of the dispersive solvent. The mis-cibility of the disperser solvent with the organic phase (ex-traction solvent) and the aqueous phase (sample solution) isthe main point for selection of the dispersive solvent in orderto achieve high EF and ER values. Methanol, acetone, ace-tonitrile, n-propanol, DMF, and DMSO, all possessing thisproperty, were investigated. n-Propanol, as dispersive sol-vent, showed the maximum extraction efficiency. The resultsobtained for the others were nearly the same. n-Propanolas well as methanol, acetone, and acetonitrile were rejecteddue to the fact that their chromatographic peaks are partiallyor completely overlapped by some analyte peaks. DMF andDMSO showed the same extraction efficiencies with cleanchromatograms. Regarding that most drugs are soluble inDMF, it was selected as the dispersive solvent.

3.3 Optimization of volume of dispersive and

extraction solvents

Volumes of organic extractant, disperser, and sedimentedphase are critical parameters of important effects on the ex-

traction efficiency and EF. The volumes of extractant anddispersive solvents were simultaneously varied in order toobtain 10 ± 1 �L-sedimented phases. The results obtainedshow that 25 and 100 �L were the optimum volumes forthe extraction and dispersive solvents, respectively (Fig. 1).It should be noted that when low volumes of extraction anddispersive solvents were used, extraction of analytes was dis-turbed due to incomplete dispersion of the organic solventsin aqueous samples. On the other hand, when high volumesof the organic solvents were used, polarity of aqueous sampledecreased due to dissolution of dispersive and extraction sol-vents in the aqueous phase that leads to a decrease in partitioncoefficients of analytes and extraction efficiencies.

3.4 Salting-out effect

In most cases, addition of a salt plays an important role inthe conventional extraction procedures. By increasing ionicstrength of aqueous sample, solubility of a non-polar or semi-polar analyte in the aqueous phase is decreased and moreanalyte molecules are transferred into the organic phase. Soextraction recovery is often increased in the presence of asalt. But this does not mean that the analytical signal willalso be enhanced. It should be noted that by adding a salt,two factors preventing analytical signal from improving areincreased: (1) sedimented phase volume that leads to a de-crease in EF and (2) viscosity of aqueous phase that leads toa decrease in diffusion coefficient. To assess the effect of saltaddition, some experiments were performed by adding dif-ferent concentrations of NaCl (0–30%, w/v). According to theobtained data (not presented in this paper), salt concentration



had not a significant effect on the analytical signal. Therefore,salt addition was not used in the subsequent experiments.

3.5 Optimization of aqueous phase volume and

centrifuging rate and time

For investigating the effect of aqueous phase volume on theDLLME performance, 2.5, 5, 7.5, and 10-mL volumes con-taining constant amounts of the analytes were tested. Theresults showed that analytical signal was nearly constant for2.5 and 5 mL and started to decrease from 7.5 on. It is notedthat the volumes of extraction and dispersive solvents werevaried proportionally so that the volume of sedimented phasevolume remained 10 �L in all cases. Therefore by increas-ing the volume of aqueous phase, the ratio of organic- toaqueous-phase volume is decreased and hence extraction ef-ficiency and analytical signals are reduced too. It seems thatthis effect is critical for high volumes of 7.5 and 10 �L.So, 5 mL was selected as the suitable volume of aqueousphase.

To study the effect of centrifugation speed and time, twoseries of experiments were carried out. In one series, a con-stant centrifugation time (3 min) was selected while its speedwas varied (1500, 3000, 4500, and 6000 rpm). Another se-ries were performed at a constant centrifugation speed (3000rpm) while the centrifuging time was different (1, 3, 5, and10 min). From the obtained results, centrifugations time andspeed were less effective. To obtain repeatable, data they wereselected as 3 min and 3000 rpm, respectively.

3.6 Aqueous phase pH

In DLLME, as well as a traditional extraction procedure, whenthe analyte molecules contribute in an acid-base equilibrium,pH of aqueous phase becomes an important factor. It ion-izes to produce a proton (H+) and an anion or to react withH+ to form a cation. However, in this study all analytes arevolatile organic solvents that do not undergo acid-base reac-tions in aqueous medium. But the studied matrices containsome chemicals that are not soluble in deionized water. Itshould be noted that sample dissolution is a necessity forthis study so that analytes trapped in solid sample can bereleased into aqueous medium. Pharmaceuticals are solublein acidic, neutral, or alkaline media. Therefore pH studieswere performed in the presence of 1 M HCl, 0.1 M HCl,0.1 M NaOH, 0.5 M NaOH, and 1 M NaOH and the resultsobtained were compared with those of deionized water. Theresults showed that the analytical signals corresponding todeionized water and 0.1 and 1 M HCl had no significantdifferences, but by increasing NaOH concentration, the an-alytical signals gradually increased. These results indicatedthat in the case of pharmaceuticals that are soluble in analkaline solution, the standard addition method should beutilized.

3.7 Analytical features of the proposed method

Under the optimum experimental conditions, some parame-ters, namely linear range (LR), repeatability, LOD, and LOQwere determined. Good linearities (0.996 ≤ r2 ≤ 0.999) wereobserved in the range 50–160 000 �g L−1 for CT, 10–32 000�g L−1 for 1,1-DCE, 1,2-DCE, and 1,1,1-TCE, and 2–6400�g L−1 for Bz (Table 2). The LOD of the proposed method,defined as the concentration giving rise to a signal-to-noiseratio equal to 3 (S/N = 3), were 11, 3, 5, 5, and 0.6 �g L−1

and the LOQs (S/N = 10) were 40, 9, 15, 17, and 2 �g L−1

for the above-mentioned analytes, respectively. By selecting500 mg as sample size for solid pharmaceutical samples,LODs of the method were calculated as 0.11, 0.05, 0.03, 0.006,and 0.05 �g g−1 for CT, 1,1,1-TCE, 1,1-DCE, Bz, and 1,2-DCE,respectively, which are completely lower than the ICH recom-mended concentrations for the selected analytes (Table 1). Toevaluate repeatability of the method, relative standard devi-ations (RSDs) were calculated from results of six repeatedexperiments performed on standard solutions which wereprepared in the ICH recommended concentrations for all ofthe analytes except 1,1,1-TCE (8 instead of 1500 �g g−1). RSDswere between 2.5–6.1% which indicated that the method hasgood repeatability. Relatively high EFs (140–353) and satis-factory recoveries (29–71%) were achieved by this DLLMEprocedure.

3.8 Analysis of pharmaceutical samples

The proposed DLLME-GC-FID method was applied to deter-mine class 1 RS content of some pharmaceutical products.Amoxicillin three hydrate, cefepime, ceftriaxone-Na, meglu-mine compound, erythromycin, and clindamycin hydrochlo-ride were considered as samples and the method was ap-plied to them. Concentrations of all analytes were lower thancorresponding LOD of the method. To study the matrix ef-fect, added-found method was used and these pharmaceuticalproducts were spiked at three different concentration levels.On each sample, the method was repeated for three times andthe results obtained were compared with those of standardsolutions at the related concentrations that the method per-formed on them, too. The obtained relative recoveries alongwith their standard deviations are listed in Table 3. The ob-tained relative recoveries (±standard deviations) were in therange 87–121% (±1–9), 102–116% (±1–7), 86–114% (±1–5),96–115% (±1–5), and 79–103% (1–6) for CT, 1,1,1-TCE, 1,1-DCE, Bz, and 1,2-DCE, respectively, which indicate that thepresence of pharmaceuticals in concentrations much higherthan that of analytes had little effect on the enrichment anddetermination of the analytes using the developed method.Figure 2 shows the GC-FID chromatograms of blank, aque-ous standard solution and direct injection of standard solu-tion in extraction solvent. There are three peaks in the chro-matogram of blank including DMF (dispersive solvent whichwas partially extracted into extraction solvent), 1,2-DBE (ex-traction solvent), and an unknown impurity of the extraction



Table 2. Analytical features of the proposed DLLME-GC-FID method

Analyte LRa) (�g L−1) r2b) LODc) LOQd) RSD (%)e) EF ± SDf) R (%) ± SDg)

In solution In solid sample In solution In solid sample(�g L−1) (�g g−1) (�g L−1) (�g g−1)

CT 50–160 000 0.996 11 0.11 40 0.4 3.7 353 ± 3 71 ± 31,1,1-TCE 10–32 000 0.997 5 0.05 17 0.17 3.5 298 ± 4 61 ± 31,1-DCE 10–32 000 0.999 3 0.03 9 0.09 3.8 151 ± 5 31 ± 6Bz 2–6400 0.996 0.6 0.006 2 0.02 3.2 188 ± 6 39 ± 51,2-DCE 10–32 000 0.999 5 0.05 15 0.15 6.1 140 ± 5 29 ± 4

a)Linear range (LR).b)Square of correlation coefficient.c)Limit of detection (S/N = 3).d)Limit of quantification (S/N = 10).e)Relative standard deviation (C = ICH recommended concentrations for all analytes, except 1,1,1-TCE (8 instead of 1500 �g g−1), n = 6).f)Mean enrichment factor (EF) ± standard deviation (n = 3).g)Mean recovery ± standard deviation (n = 3).

solvent. By no means is the initial part of chromatogramcrowded. No additional peak is present in the chromatogramof preconcentrated aqueous solution compared to that of thesolution obtained from direct injection of standard solution

in extraction solvent, with the exception of DMF peak. Inorder to illustrate the performance of method in pharmaceu-tical samples treated by the present method, chromatogramsof unspiked and spiked samples along with related aqueous

Table 3. Study of matrix effect

Sample CT 1,1,1-TCE 1,1-DCE Bz 1,2- DCE

Added R (%) Added R (%) Added R (%) Added R (%) Added R (%)concentration ± concentration ±> concentration ± concentration ± concentration ±(�g mL−1) SDa) (�g mL−1) SDa) (�g mL−1) SDa) (�g mL−1) SDa) (�g mL−1) SDa)

Amoxicillin 0.4 103 ± 4 0.8 106 ± 1 0.8 109 ± 1 0.2 106 ± 1 0.5 94 ± 1trihydrate

0.8 104 ± 2 1.6 108 ± 1 1.6 106 ± 5 0.4 102 ± 1 1 94 ± 48 87 ± 2 16 104 ± 1 16 108 ± 2 4 107 ± 1 10 92 ± 1

Cefepime 0.4 88 ± 3 0.8 109 ± 4 0.8 104 ± 5 0.2 101 ± 2 0.5 95 ± 20.8 113 ± 9 1.6 110 ± 7 1.6 98 ± 4 0.4 102 ± 1 1 89 ± 58 115 ± 6 16 115 ± 6 16 111 ± 4 4 107 ± 1 10 97 ± 4

Ceftriaxone-Na 0.4 114 ± 1 0.8 113 ± 1 0.8 113 ± 2 0.2 115 ± 4 0.5 103 ± 50.8 121 ± 5 1.6 114 ± 3 1.6 103 ± 2 0.4 106 ± 1 1 94 ± 38 116 ± 1 16 114 ± 1 16 96 ± 2 4 106 ± 1 10 90 ± 1

Meglumine 0.4 104 ± 5 0.8 103 ± 5 0.8 103 ± 3 0.2 98 ± 1 0.5 103 ± 1compound

0.8 107 ± 1 1.6 103 ± 1 1.6 109 ± 5 0.4 108 ± 1 1 97 ± 18 106 ± 2 16 102 ± 4 16 93 ± 1 4 96 ± 1 10 79 ± 1

Erythromycin 0.4 109 ± 5 0.8 104 ± 2 0.8 86 ± 5 0.2 109 ± 1 0.5 82 ± 20.8 103 ± 6 1.6 107 ± 1 1.6 111 ± 1 0.4 103 ± 3 1 82 ± 18 107 ± 6 16 105 ± 1 16 103 ± 2 4 110 ± 2 10 87 ± 6

Clindamycin 0.4 112 ± 1 0.8 111 ± 1 0.8 95 ± 1 0.2 100 ± 1 0.5 89 ± 3hydrochloride

0.8 116 ± 1 1.6 115 ± 3 1.6 114 ± 3 0.4 108 ± 1 1 94 ± 28 120 ± 1 16 116 ± 1 16 109 ± 2 4 112 ± 5 10 87 ± 1

a)Mean recovery ± standard deviation (n = 3).



Figure 2. GC-FID chromatograms of blank(A), aqueous standard solution (50 �gmL−1 of CT, 2 �g mL−1 of Bz, and 10 �gL−1 of 1,1-DCE, 1,2-DCE, and 1,1,1-TCE) (B),and direct injection (0.5 �l) of standardsolution in extraction solvent (25 000 �gmL−1 of CT, 1000 �g mL−1 of Bz, and 5000�g mL−1 of 1,1-DCE, 1,2-DCE, and 1,1,1-TCE) (C). The preconcentration techniqueunder the optimized conditions was per-formed in the cases of (A) and (B), and0.5 �L of the sedimented phase was in-jected into GC. Peaks identification: 1, CT;2, 1,1,1-TCE; 3, 1,1-DCE; 4, Bz; 5, 1,2-DCE;6, 1,2-DBE impurity; 7, 1,2-DBE (extractionsolvent); and 8, DMF (dispersive solvent).

standard solution are shown in Fig. 3. In all samples, con-centrations of analytes were lower than corresponding LODdetermined for the method and ICH recommended concen-trations. On the other hand, in the unspiked samples, no peak

emerges at retention times corresponding to analytes eluted.Also, the analytical signals obtained from samples and deion-ized water spiked at the same concentrations are comparableindicating that there is no significant matrix effect.

Figure 3. Typical chromatograms ofaqueous standard solution (A), blankof meglumine compound (B), spikedmeglumine compound (C), blank of ery-thromycin (D), spiked erythromycin (E),blank of clindamycin hydrochloride (F),and spiked clindamycin hydrochloride(G). Peak identification: 1, CT (0.4 �gmL−1); 2, 1,1,1-TCE (0.8 �g mL−1); 3, 1,1-DCE (0.8 �g mL−1); 4, Bz (0.2 �g mL−1);5, 1,2-DCE (0.5 �g mL−1); 6, 1,2-DBE im-purity ; 7, 1,2-DBE (extraction solvent);and 8, DMF (dispersive solvent).



Table 4. Comparison of the proposed method with other methods used in determination of class 1 RSs in pharmaceuticals

Sample Sample Analyte LRa) RSDb) LODc) LOQd) Sample Method Referenceweight (�g L−1) (%) (�g L−1) (�g L−1) preparation(mg) time (min)

- - CT 63–3090 13 42 130 30 Head space- [32]1,1-DCE - - - - program1,1,1-TCE - - - - temperatureBz 34.6–1700 7.2 80 230 vaporization-1,2-DCE - - - - fast GC-mass

spectrometryPentoxyfilline - CT 100–6000 4.6 37 - - Purge and trap [33]

1,1-DCE 800–160 000 4.5 2 - GC-FID1,1,1-TCE - - - -Bz 40–35 000 4.2 20 -1,2-DCE 50–110 000 4 10 -

Promethazine 100 CT 6 45 - 10 Static [34]1,1-DCE 7.3 7.4 - headspace-1,1,1-TCE 6 18.8 - GC-FID-massBz 2.4 4.8 - spectrometry1,2-DCE 1.6 30 -

Amoxicillin sodium 200 CT 2080–104 000 - 690 2420 45 Headspace- [35]clavulanate 1,1-DCE 3960–198 000 - 160 550 GC-FIDpotassium 1,1,1-TCE 720–360 000 - 480 1680clellulose Bz 1360–68 000 - 10 40microcrytallisatemixture

1,2-DCE 960–48 000 - 100 330

Antibiotics 250 CT 160–100 000 1.2 10 - 45 Headspace- [36]1,1-DCE - - - - GC-FID1,1,1-TCE - - - -Bz 160–100 000 0.8 1.2 -1,2-DCE - - - -

Pharmaceuticals 500 CT 50–160 000 3.7 11 40 5 DLLME-GC- Present method1,1-DCE 10–32 000 3.8 3 9 FID1,1,1-TCE 10–32 000 3.5 5 17Bz 2–6400 3.2 0.6 21,2-DCE 10–32 000 6.1 5 15

a)Linear range (LR).b)Relative standard deviation.c)Limit of detection.d)Limit of quantification.

3.9 Comparison of the proposed DLLME-GC-FID

method with other methods

Some analytical features (LOD, LOQ, RSD, LR, and anal-ysis time) of the present method and those of methodsreported in literature for extraction and determination ofclass 1 RSs in pharmaceutical products are summarized inTable 4. These data reveal that the presented method haslow LODs and LOQs compared to the others. LRs of theproposed method are considerably broad. Sample prepara-tion time is short, i.e. lower than 5 min. Also, RSDs ofthe method are better than or comparable with those ofthe reported methods. It can be concluded that the pro-posed method can be used as an alternative method forUSP method in determination of class 1 RSs in pharma-ceuticals.

4 Conclusions

In this study, a rapid, simple, sensitive, and environmentallyfriendly approach based on the DLLME method has been pre-sented for the simultaneous determination of class 1 RSs inpharmaceutical products. The proposed method is the firstreported application of a DLLME procedure in determina-tion of analytes that are more volatile than DLLME solvents.DMF and 1,2-DBE were used as dispersive and extraction sol-vents, respectively. This extraction system has been employedfor fast and effective preconcentration of analytes with highEFs and satisfactory ERs. The results demonstrated that theproposed DLLME-GC-FID method has good reproducibility,wide LR, low LODs, and LOQs, and short analysis time. Thesatisfactory results obtained proved that this method can bea suitable alternative to previously reported methods.



The authors thank the Research Council of University ofTabriz for financial support.

The authors have declared no conflict of interest.

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