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Some applications of state-of-the-art capillary gas chromatography in the pharmaceutical industry

P. Sandra* Laboratory of Organic Chemistry, Ghent University, Krijgslaan 281, B-9000 Gent, Belgium, and Research Institute for Chromatography, Kennedypark 20, B-8500 Kortrijk, Belgium F. David Research Institute for Chromatography, Kennedypark 20, B-8500 Kortrijk, Belgium R. Szucs Pzer Global Research and Development, Ramsgate Road, Sandwich, Kent CT13 9NJ, United Kingdom

Some challenging pharmaceutical applications performed with state-of-the-art capillary gas chromatography (CGC) are presented. For assay and purity determinations cool on-column (COC) injection is the preferred injection technique. A fast CGC headspace method is described to determine residual solvents in pharmaceutical products. Ethylene oxide and ethylene chlorohydrin, originating from the sterilisation of packaging materials, were measured in pharmaceutical formulations in a fully automated sequence with excellent retention time and peakarea reproducibility. # 2002 Published by Elsevier Science B.V. All rights reserved.
Keywords: Assay and purity determination; Capillary gas chromatography; Ethylene chlorohydrin; Ethylene oxide; Figures of merit; Solvent residues

1. Introduction
In comparison to liquid chromatography (LC), gas chromatography (GC) is less frequently used in the pharmaceutical industry for assay and purity determinations. The main
*Corresponding author. Tel.: +32 (0)56 204031; Fax: +32 (0)56 204859. E-mail: pat.sandra@richrom.com

reason for this is that most pharmaceuticals possess rather polar and thermolabile characteristics and can be ionic in nature. In the past, derivatization to make the products volatile to allow GC analysis was often applied. But this approach, in this era of fast synthetic strategies, such as combinatorial chemistry, is seldom used nowadays on the one hand, because errors can easily be introduced in this time-consuming complementary step, decreasing ruggedness and throughput on the other hand, because of the high performance of modern LC and the electrodriven separation methods for the analysis of polar, thermolabile and ionic compounds. The application of GC in a pharmaceutical quality assurance/quality control (QA/QC) laboratory is therefore mainly restricted to the determination of organic volatile synthesis intermediates or active substances in the nal formulation (assay determination) or for the analysis of organic volatile impurities (OVIs) e.g. in starting materials for drug synthesis including solvents, nalized drugs and formulations, and packaging material. In recent years, chiral CGC has also received attention for the determination of the enantiomeric excess of volatile intermediates or nal drugs. However, CGC often is a method of choice in the pharmaceutical research and
# 2002 Published by Elsevier Science B.V. All rights reserved.

0165-9936/02/$ - see front matter PII: S0165-9936(02)00803-8

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development laboratories e.g. in pharmacokinetic studies and for identication of metabolites and impurities. Appreciated features of GC, especially CGC, include the very high resolving power, the availability of a sensitive universal detector (ame ionisation detection, FID) and the possibility of applying mass spectroscopic (MS) detection (CGC-MS) for both identication and quantication. Because of the possibility to apply electron impact ionisation, CGCMS is much more powerful than LC-MS in identifying unknowns. GC methods are routinely applied in the pharmaceutical QA/QC laboratory and the most important can be found in the European and United States Pharmacopoeia. Neophytes in the eld should also consult the Guidelines of the International Conference on Harmonisation [1], and, more especially, guidelines Q3A and Q3B for impurities in new drug products, and guideline Q3C for residual solvents. It is not the aim of this article to give an overview of applied GC methods, but rather to present some specic pharmaceutical applications in which the recent developments in CGC have been implemented and also to highlight some new trends. In recent years, several important new developments have been made in CGC and as well as in the chromatographic process itself (electronic pneumatic control, improved oven temperature stability, high column quality, etc.) and in the surrounding or hyphenated tools (thermal desorption, programmed temperature vaporizing injection, time-of-ight MS in the low- or high-resolution mode, comprehensive GCxGC . . . to mention only a few). Emphasis here is placed on the more fundamental aspects of CGC that lead to reliable and rugged data fully complying with the stringent validation requirements of the pharmaceutical industry.

improved by the introduction of: electronic pneumatic control (EPC) of the carrier gas; ovens with reliable and reproducible temperature proles; and, capillary columns properly deactivated and coated with dedicated stationary phases. Concepts, such as method translation [2] and retention time locking (RTL) [3], could be realized through these developments. With method translation software (MTS), GC conditions from a standard operating procedure can be translated to a high-speed or high-throughput method. The application of MTS will be illustrated further. Retention-time reproducibility is also outstanding nowadays, and libraries can be created with locked retention times of impurities, residual solvents, etc. RTL strongly improves the ruggedness of CGC methods.

3. Assay determination and purity determination at the 0.1% level

In both assay determination (measuring the concentration of the active solute as pure chemical or in a given formulation) and purity determination (the qualitative and quantitative determination of all impurities larger than 0.1% relative to the active solute), sample introduction (i.e. the injector choice) and detector stability are of utmost importance. COC injection is by far the best injection technique for these applications because it eliminates sample degradation as a result of thermal stress and sample discrimination. As a rule of thumb, one can state that, if the sample cannot be analysed using COC injection, the compound is not GC amenable and LC should be used. Applying COC, method reproducibility can be better than 0.5%. However, the choice of solvent is important. The solvent should be compatible with the stationary phase; otherwise, a retention gap should be connected to the analytical column. For a detailed description of cool on-column injection and solvent selection we refer the reader to the literature [4]. For purity determination, the sample capacity of the column should also be considered. In order to detect 0.1% of a solute relative to the

2. Developments related to the chromatographic process

Both the kinetic and thermodynamic aspects of the GC process could be signicantly

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active compound, a relatively high amount (micrograms) of the main compound has to be introduced on the capillary column. This often leads to an overloaded (fronting) peak for the main compound and decreased resolution. Moreover, if COC (and also splitless) injection is used, the peaks eluting just before the main compound will be distorted as a result of band broadening (reversed solvent effect). The use of wide-bore and/or thick-lm columns can partially overcome this problem. Often a compromise has to be found between sample capacity and resolution. This is illustrated in Fig. 1, which shows the purity check of a pharmaceutical product. The analysis is performed on an Agilent 6890 GC using a 25 m0.32 mm i.d. column coated with 0.52 mm 95% methyl5% phenyl silicone phase (HP-5). The sample is dissolved in cyclohexane, and 1 mL of a 0.1% solution is injected cool on-column. The injector is programmed in the oven-track mode (inlet temperature follows oven temperature). Carrier gas was helium at 10 psi inlet pressure. The oven was programmed from 50  C (0 min.) to 280  C at 10  C/min. Detection was done by FID. The chromatogram shows several impurities. The peaks at 6.636 min., 8.017 min., 10.619 min. and 11.828 min. are added impurities at the 0.1% (w/w) concentration level relative to the main compound (9.514 min.), showing that the method fully meets the sensitivity requirements. However, it is interesting to

observe that the peak of a small impurity (marked with an asterisk), eluting just before the main compound, is distorted because of the proximity of the large peak of the main product that behaves as a solvent. Nevertheless, this did not impair its quantitation (r2=0.9992 in the 0.011% calibration range). Optical purity of pharmaceuticals is normally performed by LC, packed column supercritical uid chromatography (pSFC) or capillary electrophoresis (CE). LC is the method of choice because it combines good enantioselectivity, high sample capacity, up-scaling features to preparative LC and wide availability of LC instrumentation. The use of chiral CE is presently gaining momentum in the pharmaceutical industry for the determination of the enantiomeric excess of chiral drugs. But, for volatile compounds lacking a chromophore, CGC can offer a good analytical alternative. The best chiral stationary phases (CSPs) in CGC are the diamide phase Chiralsil-Val and the cyclodextrin phases. As an illustration, Fig. 2 shows the separation of an intermediate of the enantioselective synthesis of vitamin D analogues. 1 ml of the sample solubilized at 0.05% in n-hexane/ ethyl acetate (9/1) was injected via COC on a 25 mL250 mm i.d. fused silica capillary column coated with 0.25 mm 2,3-di-O-methyl-6-OTBDMS b-cyclodextrin in 50% OV-1701. The column was operated at 50  C during injection (1 min.) and then programmed at 40  C/min. to

Fig. 1. Analysis of impurities in a pharmaceutical product by cool on-column (COC) injection capillary GC-FID. * Impurity eluting before the main product. Conditions : see text.

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125  C. Hydrogen was the carrier gas at 10 psi. Determination of the enantiomeric excess gave 99.16% R with an RSD% for n=6 of 0.14.

4. Determination of residues at the ppm (mg/kg or mg/l) level

The pharmaceutical industry can nowadays substantially accelerate the development of new active compounds. In parallel with the increased number of candidates in early and full development, faster separation techniques are mandatory. This is true for not only the more frequently applied liquid-based separation techniques [5] but also GC. One of the most important applications of GC in the pharmaceutical industry is the analysis of residual solvents throughout the various stages of drug development. Although the theoretical description and illustration of the possibilities of fast GC were performed 40 years ago [6], its introduction in routine laboratories was possible only after recent advances in instrumentation and in column technology. Indeed, narrow-bore columns with i.d.s smaller than 200 mm are mandatory for fast, high-resolution GC, and dedicated instrumentation and software to help

translate standard operating conditions on conventional columns are required. Several applications, based on the principle of method translation, have already appeared in literature [7]. The situation is less straightforward when practical implementation of fast residual solvent analysis is considered. Standard solvent residue analysis is performed using static headspace injection. The sample is, therefore, dissolved in a suitable solvent and enclosed inside a headspace vial for equilibration at elevated temperatures. Afterwards, the headspace is transferred towards the analytical instrument, avoiding the injection of non-volatile drug substances. In our laboratories, two methodologies are used for fast, reliable analysis of residual solvents in pharmaceutical formulations. Both methods use standard headspace-GC instrumentation. The rst (generic) method is used for identication of unknown solvent residues, whereas the second method is used for fast throughput and quantication of samples already resolved with the generic method. The generic method is illustrated with the analysis of a mixture with 20 solvents frequently used in the synthesis of drugs (Fig. 3A). A stock solution was prepared in N,N-dimethylacetamide

Fig. 2. Chiral separation of a pharmaceutical product on a fused silica capillary column coated with 2,3-di-O-methyl-6-OTBDMS b-cyclodextrin.

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Fig. 3. Static headspace-capillary GC analysis of residual solvents in pharmaceutical products. A: Generic separation on a 30 m320 mm i.d.1.8 mm 624 column. B: Translated fast method on a 25 m150 mm i.d.0.84 mm 624 column. C: Fast screening method on a 25 m150 mm i.d.0.84 mm 624 column using a modied temperature program. Peaks: 1. methanol; 2. n-pentane; 3. ethanol; 4. acetone; 5. acetonitrile; 6. isopropanol; 7. dichloromethane; 8. t-butanol; 9. n-hexane; 10. isopropyl ether; 11. 2-butanone; 12. ethyl acetate; 13. tetrahydrofuran; 14. chloroform; 15. cyclohexane; 16. 2-methoxyethanol; 17. n-heptane; 18. methyl isobutyl ketone; 19. toluene; 20. n-butyl acetate.

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(DMAC) at the level of 1% w/w for each solvent. Analyses were performed using 5 mL samples of a 1:100 dilution of the standard mixture in DMAC. Samples were transferred to 20 mL headspace vials, which were capped and thermostated at 85  C for 10 min. in the autosampler oven. In order to accelerate solvent evaporation, samples were shaken automatically in the oven at the highest agitation setting. Afterwards, the headspace vial was pressurised for 0.5 min. using helium at 12 psi. Loop-ll and equilibration times were set at 0.05 min. and 0.3 min., respectively. Transfer-line and loop temperature were maintained at 140  C. The sampler settings, with the exception of transfer-line pressure, which was chosen as function of column dimensions, were the same in the generic and high-throughput methods. A column, coated with a thick lm of the stationary phase 624 (6% cyanopropylphenyl-94% dimethylpolysiloxane), as recommended in the Pharmacopoeia [8,9] and commercially available from Varian-Chrompack, was installed in an GC (Agilent 6890) equipped with a headspace sampler (Agilent 7694). The chromatographic conditions are listed in Table 1. The slow rst oven heating rate is characteristic for a generic method and is required to achieve maximal peak capacity, i.e. to resolve as much of the solvents as possible in a single run. Consequently, samples with unknown composition can be identied based on unique retention times without the necessity

for more complicated hyphenated detection techniques and an RTL database can be created. When oven cool down time is included, sampleto-sample analysis time (cycle time) runs to 43 min. in total. As is clear from Fig. 3A, the test mixture includes a number of crucial sections. The rst involves the cluster with closely eluting solvents: acetone, acetonitrile, iso-propanol, dichloromethane and t-butanol (peaks 48). Two critical solvent pairs are also present in the chromatogram. The rst pair comprises 2-butanone and ethyl acetate (peaks 11 and 12). The second pair is tetrahydrofuran (THF) and chloroform (peaks 13 and 14). Sample polarity ranges from the highly polar alcohols, methanol and ethanol, to the apolar n-alkanes (n-C5n-C7). The method is typically used to cover the concentration range between 0.001% w/w and 1% w/w. Quantication is performed relative to the 0.01% w/w level. As a result of these relatively high concentration levels, split injection is able to realise enough sensitivity for the complete mixture. Split ow, as measured at the instrument split vent, was 13.5 mL/min. Conditions of the generic method were translated with the MTS software, which should theoretically give the same resolution as the original analysis for a narrow-bore column coated with the same stationary phase (Table 1). On special request, the column was prepared by Varian-Chrompack. The transfer-line pressure was set at 70 psi during injection (split ow

Table 1 Operating conditions as calculated by the method translation software Column length internal diameter lm thickness inlet P at 40  C transfer line P Oven program initial ramp 1 ramp 2 nal 40  C, 5 min. 90  C at 2  C/min. 225  C at 30  C/min. 225  C, 2 min. 40  C, 2.25 min. 90  C at 66.58  C/min. 225  C at 66.58  C/min. 225  C, 0.90 min. Conventional 30 m 320 mm 1.8 mm 6.20 psi 20 psi Narrow bore 25 m 150 mm 0.84 mm 49.01 psi 20 psi

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105 mL/min.) and afterwards ramped to 85 psi at 150 psi/min. The resulting chromatogram is shown in Fig. 3B. Complete separation is achieved within 16 min. with a total cycle time of ca. 21 min. Compared to the standard chromatogram in Fig. 3A, the global chromatographic prole is maintained. Limits of quantication (LOQ), dened as 10 times the signal-to-noise ratio of an individual peak, were the same as on the conventional column. The number of residual solvents to be determined in a pharmaceutical intermediate or formulation is usually restricted to between one and ve components. As a result, the largest part of the available peak capacity is not used, resulting in a signicant over-resolution in the nal chromatogram. In order to minimise the effect of over-resolution, the analysis was further accelerated by incorporating a ballistic oven-heating ramp to achieve fast elution of the higher molecular-weight solvents (Fig. 3C). The rst part of the program was the same as for Fig. 3B but ballistic heating (120  C/ min. to 225  C) was applied at 4.5 min. The nal temperature was held for 1 min. The analysis time is reduced to 7 min. and the cycle time to 12 min. Some gures of merit are listed in Table 2. An even more challenging GC analysis concerns the determination of ethylene oxide and ethylene chlorohydrin (2-chloroethanol) in pharmaceutical products. For quality control, after sterilisation of the packaging materials, it is necessary to monitor possible migration of these toxic compounds into the pharmaceutical formulation itself. Normally, these compounds are analysed by two different GC methods applying different analytical conditions.
Table 2 Figures of merit for the fast headspace method (n=6) No. Solvent Sensitivity LOQ %w/w 1 6 7 9 12 14 19 methanol iso-propanol dichloromethane n-hexane ethyl acetate chloroform toluene 0.0020 0.0022 0.0028 0.0001 0.0010 0.0140 0.0004 RSD at LOQ 1.7 5.3 8.9 5.7 7.0 6.8 8.0

For viscous pharmaceutical products, ethylene oxide is analysed after liquid extraction with acetonitrile and a detection limit of 1 ppm is required. Because the solvent elutes after the target compound, split injection has to be applied in CGC; otherwise, there is peak splitting, which makes quantication erratic. In order to reach the specied detection limit, a low split ratio is mandatory. For the analysis of ethylene chlorohydrin, an acetonitrile extraction is also used and the required detection limit is 20 ppm. However, ethylene chlorohydrin elutes after acetonitrile and therefore a different split ratio and temperature program are required. Using state-ofthe-art capillary GC instrumentation with EPC on the carrier gas and detector gases, it is possible to analyse both products in the same sample in a fully automated sequence by applying different chromatographic conditions, including a different column ow, a different split ratio, a different oven-temperature program and different detector-gas ows. An aqueous pharmaceutical product used for eye surgery was spiked with 5 ppm ethylene oxide and 25 ppm ethylene chlorohydrin. 1 g product was extracted with 2 mL acetonitrile using vortex mixing. The analyses were carried out on a GC (Agilent 6890) equipped with automated split injection (Agilent 7673 autosampler). Both compounds can be analysed on a polar WAX column. The instrumental conguration and analytical conditions are summarized in Table 3. The conditions used for ethylene oxide are listed under Experimental conditions A, and those for ethylene chlorohydrin under

Repeatability RT RSD 0.03 0.08 0.08 0.09 0.10 0.06 0.02 Area RSD 0.60 0.52 0.30 0.24 0.25 0.46 0.43

Linearity Correlation 0.99995 0.99991 0.99996 0.99999 0.99996 0.99989 0.99989 Intercept 0.340549 0.058522 0.083321 2.233390 0.083898 0.013416 0.180075

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Experimental conditions B. For all analyses, the same inlet and detector temperatures were used. For ethylene oxide, a 1/10 split ratio was applied, while the split ratio was 1/25 for ethylene chlorohydrin. In addition, different column ows, oven temperature programs and detector gas ows were used. Both sets of conditions were saved under a different method name. The spiked sample was then analysed in a sequence of 45 analyses, comprising nine runs with method A (ethylene oxide), nine with B (ethylene chlorohydrin), nine with A, nine with B and, nally, nine with A. The optimum detector gas ows were rst evaluated for both compounds. This could be performed using a sequence of different methods, each with different detector ows. It was interesting to observe that a different optimum hydrogen ow was obtained for the two compounds, namely 25 mL/min. for ethylene oxide and 35 mL/min. for ethylene chlorohydrin. A typical chromatogram for the analysis of ethylene oxide, using experimental conditions A, is shown in Fig. 4A and for the analysis of ethylene chlorohydrin, using experimental conditions B, in Fig. 4B.

From these chromatograms, it is clear that sufcient sensitivity is achieved. The analysis time is relatively short (10 min. per analysis), and both analyses can be performed within 30 min., including oven cool down and equilibration time. The reproducibility of retention times and peak area for both compounds was measured for the sequence of 45 analyses. The results are shown in Fig. 5A for retention times and Fig. 5B for absolute area. The overall standard deviation on the retention times is smaller than 0.002 min. (< 0.05% RSD). This reproducibility is remarkable, since the column ow and the oven temperature are changed for each set of nine runs. The same observation can be made for the reproducibility of the peak areas. For ethylene oxide, the relative standard deviation is smaller than 1% and for ethylene chlorohydrin the relative standard deviation is smaller than 1.5%. These RSDs are measured for the total number of analyses performed and, although different methods with different split ratios, column ows, oven temperatures and detector gas ows

Table 3 Chromatographic system and experimental conditions for the determination of ethylene oxide and ethylene chlorohydrin Chromatographic system Injector Liner Detector Column Experimental conditions A Inlet temperature Injection volume Split ratio Carrier gas Head pressure Flow Oven temperature Detector temperature Detector gases Experimental conditions B Inlet temperature Injection volume Split ratio Carrier gas Head pressure Flow Oven temperature Detector temperature Detector gases split/splitless split/splitless liner (4 mm i.d. with glass wool plug) FID 60 m0.53 mm i.d.1 mm HP-WAX (Agilent Technologies) 250  C 1 ml 1/10 Hydrogen 3.6 psi at 50  C- constant ow mode 5.2 mL/min. 50  C, 5  C/min. to 75  C, 25  C/min. to 200  C 250  C Hydrogen : 25 mL/min.; Air : 400 mL/min.; Helium : 30 mL/min. 250  C 1 ml 1/25 Hydrogen 7.2 psi at 150  C- constant ow mode 7.5 mL/min. 150  C, 5  C/min. to 200  C 250  C Hydrogen : 35 mL/min.; Air : 400 mL/min.; Helium : 30 mL/min.

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were used, these values are not signicantly larger than the RSDs for each set of nine runs. This reproducibility is obtained thanks to the fact that all pneumatics are electronically controlled, resulting in very high accuracy and reproducibility of instrumental parameters. The analysis

of ethylene oxide and ethylene chlorohydrin can thus be performed with the same instrumental set-up in an unattended sequence, whereby it is not even necessary to recalibrate the instrument for each set of measurements. This signicantly improved productivity and cost.

Fig. 4. A: Analysis of 5 ppm ethylene oxide (ETO) in a pharmaceutical formulation using optimised conditions A (see text and Table 3); B: Analysis of 25 ppm ethylene chlorohydrin (ECH) in a pharmaceutical formulation using optimised conditions B (see text and Table 3).

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of the technique for QA/QC in the pharmaceutical industry. Developments, such as EPC, narrow-bore columns and method translation and retention-time locking, should be fully exploited in this era of fast synthetic strategies.

References
[1] Guidelines of the International Conference on Harmonisation (http://www.ifpma.org/ich1.html). [2] B.D. Quimby, V. Giarrocco, M.S. Klee, Hewlett-Packard Application Note 228-294, (February 1995), Publication Number (43) 59635190E. [3] V. Giarrocco, B. Quimby and M.S. Klee, Agilent Technologies Application Note 228373, (March 1997), Publication Number 59657673E. [4] K. Grob, On-Column Injection in Capillary Gas Chromatography: Basic Technique, Retention Gaps, Solvent Effects, Huethig Verlag, Heidelberg, Germany, 1987. [5] P. Sandra, LC.GC Europe, Supplement Guide to LC-MS, December 2001, p. 8. [6] D.H. Desty, A. Goldup, W.T. Swanton, Gas Chromatography, in: N. Brenner, J.E. Callen, M.D. Weis (Editors), Academic Press, London, 1962, p. 105. [7] F. David, D.R. Gere, F. Scanlan, P. Sandra, J. Chromatogr. A 842 (1999) 309. [8] United States Pharmacopoeia, Organic Volatile Impurities <467 > , Methods I, IV and V, 2002. [9] European Pharmacopoeia, Section V.3.3.9.-2 Residual Solvents, System A, 2002. Pat Sandra is Professor of Separation Sciences at Gent University, Belgium, and at Stellenbosch University, South Africa. He is also Director of the Research Institute for Chromatography, Kortrijk, Belgium. Frank David is R&D manager at the Research Institute for Chromatography, Kortrijk, Belgium. Roman Szucs is Principal Scientist at the Pzer Global Research and Development Centre in Sandwich, United Kingdom.

Fig. 5. A: Variation of retention times of ethylene oxide (ETO) and ethylene chlorohydrin (ECH) as a function of analysis number; B: Variation of peak areas of ethylene oxide (ETO) and ethylene chlorohydrin (ECH) as a function of analysis number.

5. Summary
Recent advances in CGC column technology and instrumentation have tremendously improved the performance and the ruggedness