Short papers in pharmaceutical analysis
Identifieur interne : 002136 ( Istex/Corpus ); précédent : 002135; suivant : 002137Short papers in pharmaceutical analysis
Auteurs : Chris J. Goodwin ; K. D. Altria ; S. D. Filbey ; A. Scott ; P. R. Vojvodic ; N. H. Anderson ; B. J. Clark ; Alison E. Bretnall ; Thomas Cowen ; Rachel Howling ; George Hutchinson ; K. M. Sereda ; T. C. Hardman ; M. R. Dilloway ; A. F. Lant ; Ian D. Smith ; Paul D. Blackler ; David G. WatersSource :
- Analytical Proceedings [ 0144-557X ] ; 1993.
English descriptors
- Teeft :
- Active metabolites, Aliquot, Aliquot condition, Analogue, Analytical methods, Analytical proceedings, Analytical rationalization, Aqueous chloride, Aqueous component, Aqueous potassium, Aqueous potassium chloride, Assay, Atenolol, Atenolol peak, Atenolol solution, Baseline resolution, Bromide, Bromide peak, Capillary electrophoresis, Carboxylic acid groups, Careful control, Chemical structures, Chiral, Chiral centre, Chiral recognition sites, Chloride, Chlorine atoms, Chromatogr, Chromatogram, Chromatograph, Chromatographic conditions, Chromatographic parameters, Chromatographic separation, Ciprofibrate, Critical separation, Cyclopropane ring, Dichloro analogue, Drug content, Ester, Ester linkage, Ethyl esters, Extensive metabolizers, Flow rate, Good performance, Hplc, Hplc system, Human urine, Impurity, Inhibitor, Injection volume, Inorganic modifier, Inorganic salts, Ionic bromide, Ionic chloride, Large number, Linearity, Liquid chromatography, Market place, Methyl, Methyl ester, Methyl group, Microcrystalline cellulose triacetate, Mobile phase, Mobile phase composition, Mobile phases, Nominal concentrations, Organic modifier, Parent compound, Particular separation, Peak area ratio, Peak areas, Pentafluorobenzyl bromide, Pharmaceutical, Pharmaceutical analysis, Pharmaceutical products, Phenyl, Phenyl ring, Poor metabolizers, Potassium, Potassium chloride, Potential impurities, Process optimization, Quantitative determination, Ramiprilat, Reagent, Retention time, Retention times, Rnin, Sample preparation, Sample solutions, Separation conditions, September, Sharp peak, Silica, Sodium dihydrogenorthophosphate, Stability batches, Standard curves, Standard deviations, Standard solution, Standard solutions, Stationary phase, Sulfate, Sumatriptan, Sumatriptan cmp3, Sumatriptan content, Total chloride, Total halides, Triacetate, Typical chromatograms, Urine, Urine extracts.
Url:
DOI: 10.1039/AP9933000361
Links to Exploration step
ISTEX:87BC31C4076810D9EF32DC83ECCADA8A4FFEF340Le document en format XML
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J.</forename><surname>Clark</surname></persName></author><author xml:id="author-0007"><persName><forename type="first">Alison E.</forename><surname>Bretnall</surname></persName></author><author xml:id="author-0008"><persName><forename type="first">Thomas</forename><surname>Cowen</surname></persName></author><author xml:id="author-0009"><persName><forename type="first">Rachel</forename><surname>Howling</surname></persName></author><author xml:id="author-0010"><persName><forename type="first">George</forename><surname>Hutchinson</surname></persName></author><author xml:id="author-0011"><persName><forename type="first">K. M.</forename><surname>Sereda</surname></persName></author><author xml:id="author-0012"><persName><forename type="first">T. C.</forename><surname>Hardman</surname></persName></author><author xml:id="author-0013"><persName><forename type="first">M. R.</forename><surname>Dilloway</surname></persName></author><author xml:id="author-0014"><persName><forename type="first">A. F.</forename><surname>Lant</surname></persName></author><author xml:id="author-0015"><persName><forename type="first">Ian D.</forename><surname>Smith</surname></persName></author><author xml:id="author-0016"><persName><forename type="first">Paul D.</forename><surname>Blackler</surname></persName></author><author xml:id="author-0017"><persName><forename type="first">David G.</forename><surname>Waters</surname></persName></author><idno type="istex">87BC31C4076810D9EF32DC83ECCADA8A4FFEF340</idno><idno type="ark">ark:/67375/QHD-JMTF2065-V</idno><idno type="DOI">10.1039/AP9933000361</idno><idno type="ms-id">AP9933000361</idno></analytic><monogr><title level="j">Analytical Proceedings</title><title level="j" type="abbrev">Anal. Proc.</title><idno type="pISSN">0144-557X</idno><idno type="coden">ANPRDI</idno><idno type="RSC-sercode">AP</idno><imprint><publisher>The Royal Society of Chemistry.</publisher><date type="published" when="1993"></date><biblScope unit="issue">9</biblScope><biblScope unit="page" from="361">361</biblScope><biblScope unit="page" to="374">374</biblScope></imprint></monogr></biblStruct></sourceDesc></fileDesc><profileDesc><creation><date>1993</date></creation><langUsage><language ident="en">en</language></langUsage></profileDesc><revisionDesc><change when="1993">Published</change></revisionDesc></teiHeader></istex:fulltextTEI><json:item><extension>txt</extension><original>false</original><mimetype>text/plain</mimetype><uri>https://api.istex.fr/ark:/67375/QHD-JMTF2065-V/fulltext.txt</uri></json:item></fulltext><metadata><istex:metadataXml wicri:clean="corpus rsc-journals not found" wicri:toSee="no header"><istex:xmlDeclaration>version="1.0" encoding="UTF-8"</istex:xmlDeclaration><istex:document><article type="ART" xsi:schemaLocation="http://www.rsc.org/schema/rscart38 http://www.rsc.org/schema/rscart38/rscart38.xsd" dtd="RSCART3.8"><metainfo><articletype code="ART" pubmedForm="research-article" headingForm="Papers" group="Paper" fullForm="Paper"></articletype></metainfo><art-admin><ms-id>AP9933000361</ms-id><doi>10.1039/AP9933000361</doi></art-admin><published type="print"><journalref><link>AP</link></journalref><volumeref><link>30</link></volumeref><issueref><link>9</link></issueref><pubfront><fpage>361</fpage><lpage>374</lpage><no-of-pages>14</no-of-pages><date><year>1993</year></date></pubfront></published><published type="subsyear"><journalref><title type="abbreviated">Anal. Proc.</title><title type="full">Analytical Proceedings</title><title type="journal">Analytical Communications</title><title type="display">Analytical Communications</title><sercode>AP</sercode><publisher><orgname><nameelt>The Royal Society of Chemistry</nameelt></orgname></publisher><issn type="print">0144-557X</issn><coden>ANPRDI</coden><cpyrt>This journal is © The Royal Society of Chemistry</cpyrt></journalref><pubfront><fpage></fpage><lpage></lpage><no-of-pages></no-of-pages><date><year>1993</year></date></pubfront></published><art-front><titlegrp><title>Short papers in pharmaceutical analysis</title></titlegrp><authgrp><author><person><persname><fname>Chris J.</fname><surname>Goodwin</surname></persname></person></author><author><person><persname><fname>K. D.</fname><surname>Altria</surname></persname></person></author><author><person><persname><fname>S. D.</fname><surname>Filbey</surname></persname></person></author><author><person><persname><fname>A.</fname><surname>Scott</surname></persname></person></author><author><person><persname><fname>P. R.</fname><surname>Vojvodic</surname></persname></person></author><author><person><persname><fname>N. H.</fname><surname>Anderson</surname></persname></person></author><author><person><persname><fname>B. J.</fname><surname>Clark</surname></persname></person></author><author><person><persname><fname>Alison E.</fname><surname>Bretnall</surname></persname></person></author><author><person><persname><fname>Thomas</fname><surname>Cowen</surname></persname></person></author><author><person><persname><fname>Rachel</fname><surname>Howling</surname></persname></person></author><author><person><persname><fname>George</fname><surname>Hutchinson</surname></persname></person></author><author><person><persname><fname>K. M.</fname><surname>Sereda</surname></persname></person></author><author><person><persname><fname>T. C.</fname><surname>Hardman</surname></persname></person></author><author><person><persname><fname>M. R.</fname><surname>Dilloway</surname></persname></person></author><author><person><persname><fname>A. F.</fname><surname>Lant</surname></persname></person></author><author><person><persname><fname>Ian D.</fname><surname>Smith</surname></persname></person></author><author><person><persname><fname>Paul D.</fname><surname>Blackler</surname></persname></person></author><author><person><persname><fname>David G.</fname><surname>Waters</surname></persname></person></author></authgrp></art-front><art-body><section type="PDFExtract"><title></title><p> ANALYTICAL PROCEEDINGS, SEPTEMBER 1993, VOL 30 361 Short Papers in Pharmaceutical Analysis The following are summaries of eight of the papers presented at a Meeting of the Joint Pharmaceutical Analysis Group held on October 15th, 1992, in the Royal Pharmaceutical Society, London SEI. Analytical Rationalization Chris J. Goodwin Smith Kline Beecham Pharmaceuticals, Clarendon Road, Worthing, West Sussex BN 14 8QH From the time a new pharmaceutical product is discovered, its journey through to the market place can be as long as 15 years. During this time the drug substance will pass through many stages of development including laboratory and semi-scale synthesis, pilot plant scale-up, efficacy and toxicology testing, clinical trials and finally full-scale production. During this time, knowledge of the chemical process will grow considerably. </p><p>Opportunities to change the route of manufacture may well give rise to novel potential impurities. The analytical systems applied during this time will also evolve. These often become more complex until a step change occurs to rationalize them. This may take place when a product is relocated to another site and re-registration becomes necessary. During scale-up and process optimization a very large number of samples are analysed. The immediate reward for simplification of the analytical regime is considerable. In financial terms there will be benefits for many years at the production site; and the improved analytical lead times aid plant and development process control and project progress, speeds up the development progress, and reduces the time taken for the product to reach the market place. </p><p>The analytical rationalization process can be demonstrated using as examples the analytical system for a new antiviral drug called penciclovir. Penciclovir is the purine derivative of I used for the treatment of various herpes virus infections including Varicella Zoster, commonly known as shingles. It acts on the process of DNA replication in virally infected cells only. Penciclovir 0 A, B and C which would be very time consuming and, with the large number of samples generated during process optimiz- ation, impractical. For example, to run a full impurity profile of 15 batches, 3 days labour and instrument usage would be required. It should be remembered at this point that the existing method evolved with the process and that all the potential impurities were not known in the early stages of development. </p><p>It would be impossible to develop a method at the start to separate all the impurities eventually known in the fully developed process. A gradient HPLC method has been successfully developed which achieves baseline resolution of 16 potential impurities in a single run of 60 rnin with a 10 min equilibration time (Fig. 1) LOH 9-(4-Hydroxy-3-hydroxymethylbutyl)guanine (BRL 39123) I When the development of this product was expanded, the established existing methodology for product impurity profil- ing which had been developed along with the process, consisted of three isocratic high-performance liquid chromatography (HPLC) methods. </p><p>Each sample would be analysed on systems :-- BRL 42817 F? \ 5 % t- / BRL 46970 1 BRL 391231 $ p F BRL 42377 =. 0 - BRL 48440 BRL 4601 3 2 5. EL BRL 451 39 %. BRL 39913 BRL 46969 BRL 46771 BRL 46979 Fig. 1 Chromatogram of a BRL 39123 batch spiked with potential impurities at levels of 0.1%. The following HPLC conditions were used: Column: SPHERISORB ODS 2 (250 x 4.6 mm i.d.); eluents: A, 0.1 mol dmP3 sodium dihydrogenorthophosphate pH 3.7; B, 79% 0.1 mol dm-3 sodium dihydrogenorthophosphate pH 3.7,21% HPLC grade acetonitrile; wavelength 254 nm; flow 2 cm3 min-'. Gradient: 0-5 min, 100% A (isocratic); 5-35 rnin to 65% A and 35% B (linear); 35-50 rnin to 100% B (linear); 50-60 rnin to 100% B (isocratic); 60-65 mins to 100% A (linear); 65-70 min remaining at 100% (equilibration period)362 ANALYTICAL PROCEEDINGS, SEPTEMBER 1993, VOL 30 The method was developed using linear gradients to allow easy transfer between sites and types of equipment. </p><p>The potential impurities involved in the critical separation are shown in Fig. 2. There were two areas of critical separation. The first critical separation was achieved using: (a), careful control of eluent pH (at 3.7 to ensure the isomer BRL 46970 eluted before penciclavir I); (b), the introduction of a 5 rnin run at the start with no organic modifier; and also (c) by the use of acetonitrile and 0.1 mol dmP3 sodium dihydrogenorthophos- phate, which sharpened the peaks in comparison with the existing method that used a 0.05 mol dm-3 phosphate-meth- anol eluent. The second critical separation between BRL 39206, BRL 42222, BRL 56225 and BRL 56293 was achieved only with very 0 0 BRL 391 23 careful control of the acetonitrile content; hence the gradient is very gentle from 5 to 35 min (065% eluent B). </p><p>The gradient HPLC impurity method has been successfully used in a routine environment for 18 months. It has proved very rugged and has resulted in a 75% labour saving for the analysis of penciclovir. There are other examples of analytical rationalization in the penciclovir project. A fast liquid chromatography (LC) method was developed for one of the process intermediates where baseline resolution was achieved from the potential impurities and process reagents in a single run time of 8 min. The conditions used were as follows. Column: Jones 3 prn CI8 (50 x 4.6 mm i.d.1 + 10 mm guard column; eluent: 67% 0.05 mol dm-3 sodium dihydrogenorthophosphate; 33% HPLC BRL 42377 HO HO OH BRL 39206 b HO OH BRL 46970 OH 0 BRL 42222 I HO h CI BRL 56225 BRL 56293 Fig. </p><p>2 Potential impurities involved in critical separationsANALYTICAL PROCEEDINGS, SEPTEMBER 1993, VOL 30 grade acetonitrile; flow: 2.0 cm3 min-l; wavelength: 257 mm; typical main peak retention time 6.0 min. This method has proved to be very rugged in routine use and replaced a gas chromatography (GC) method with a 29 min run time (including reconditioning). A modified version is also used for impurity profiling. A further example of analytical rationalization was the replacement of a normal-phase HPLC method with a more rugged reversed-phase system. </p><p>The side chain intermediate was 363 baseline-resolved from its isomer. The reversed-phase method was found to be more reproducible and easier to operate. In companies where a significant number of samples are being assessed analytical rationalization is very beneficial. When the production of a new product is scaled up and the testing becomes more routine, new more streamlined analyti- cal techniques are very often required to meet different demands, not only of analytical lead times but also the type of environment in which the analysis is carried out. Quantitative Determination of Sumatriptan by Capillary Electrophoresis K. D. Altria and S. D. Filbey Pharmaceutical Analysis, Glaxo Group Research, Park Road, Ware, Hertfordshire SG 12 ODP The determination of drug content in formulated pharmaceut- ical products is performed predominantly by high-performance liquid chromatography (HPLC) as this can offer a fast and automated determination. </p><p>Capillary electrophoresis (CE) is a complementary technique that can achieve equally rapid, high- resolution separation. CE has been investigated for a number of applications within pharmaceutical analysis. 1-3 However, there has been little emphasis on demonstrating that these methods are capable of routinely determining the drug content of formulated pharmaceutical products. CE has been employed for the quantitative determination of sumatriptan [3- (2-dimethylaminoethyl)-5-( methylaminosulfonylmethyl)in- dole] levels in subcutaneous injection solutions. Sumatriptan is marketed for the treatment of migraine. </p><p>Results generated by both CE and HPLC for four batches of sumatriptan injection solutions compared well. The CE method gave good per- formance in terms of selectivity, precision, linearity and repeatability of both injection and analysis. Theory In capillary zone electrophoresis separations are achieved by the application of high voltages; ionic species migrate electro- phoretically in an applied electric field. Ions are separated according to their mobility in field solution, mobility being dependent upon the charge-to-size ratio of the ion. Separation of species with ionizable groups can be optimized largely by varying pH. Experimental Chemicals were obtained from Aldrich Ltd. (Gillingham, Dorset, UK), and water was obtained from a Millipore Q system (Watford, Hertfordshire, UK). </p><p>The quantitative work was performed on a Waters Quantum 4000 CE instrument (Watford, Hertfordshire, UK) which was connected to a Hewlett-Packard data collection system (Bracknell, Berkshire, UK). Comparative separations were achieved on a P/ACE 2000 CE instrument (Beckman, Palo Alto, CA, USA). The fused silica capillaries used in this study were purchased from both Waters and Beckman. An internal standard was employed since this had been shown to improve the repeatability of injection in CE.4 A precursor to ranitidine5 was selected (chemical structures are given in Fig. 1) as this was known to migrate before any sumatriptan related compounds. Sample and standard solu- tions were prepared to give a final aqueous concentration of 0.5 mg cm-3 of both internal standard and sumatriptan. </p><p>Results CE was used to quantify levels of sumatriptan in injection solutions, the samples selected for this purpose contained sumatriptan formulated at 12 mg cm-3 in isotonic saline solution. Currently HPLC methods are employed for the determination. Method Development Practical guidelines to the method development options for CE of pharmaceuticals have recently been published by McLaugh- lin et al. For this particular separation a low pH (pH 2.3) was selected to ensure protonation of both the analyte and related impurities. The CE separation of a synthetic test mixture of sumatrip- tan, a dimeric related impurity, and the internal standards is shown in Fig. 2. Precision of Injection Relative standard deviations (RSDs) of less than 2% can be routinely obtained for peak areas on commercial instruments. </p><p>By employing an internal standard, variability can be reduced still further with typical RSDs of below 1% being ~ b t a i n e d . ~ Replicate electropherograms for sample solutions are shown in Fig. 3; these separations indicate the consistent impurity profiles obtained throughout these studies. Both a calibration and sample solution were injected 5 times and acceptable precison for peak area and peak area ratios was obtained (Table 1). Migration time variation using the CE method, measured in H Internal standard ‘0’ Fig. 1 Chemical structures of sumatriptan and internal standard364 ANALYTICAL PROCEEDINGS, SEPTEMBER 1993, VOL 30 t 17.00 I* 13.00 1 2.00 4.00 6.00 8.00 Retention ti rn e/rn i n Fig. </p><p>2 CE separation of internal standard (A), sumatriptan related dimeric impurity (B) and sumatriptan (C). Separation conditions: 20.0 s hydrodynamic sampling, +20 kV, 214 nm, 75 ym X 60 cm fused silica capillary, sodium dihydrogenorthophosphate (25 mmol dm-3, pH 2.3 with concentrated phosphoric acid) 18.40 - 18.20 - 18.00 17.80 - - 17.40 17.20 ' 17.001 I I , 1 18.40 18.20 18.00 17.80 17.60 17.40 17.20 17.00 1 I , , 3.00 5.00 7.00 9.00 Retention ti me/rn i nutes Fig. 3 see caption to Fig. 2 CE separation of sample solutions. For separation conditions Table 1 Precision of injection peak area (number of injections = 5) RSD(%) Standard Sample solution solution Sumatriptan 0.7 0.7 IS 0.5 0.1 Peak area ratio 0.5 0.8 terms of migration time and relative migration time, was typically less than 1% RSD. </p><p>Sensitivity A limit of detection of 0.1% m/m of the sumatriptan loading (0.5 mg cmP3) was obtained (signal-to-noise ratio greater than 3). A similar detection limit of 0.1% aredarea for salicylamide impurities by CE has been reported.' This limit of detection of equivalent to 500 ppb sumatriptan in solution. Linearity The linearity of detector response between 0 and 150% of the sample concentration (0.5 mg cm-3 sumatriptan) was estab- lished. The data showed good linearity for both sumatriptan peak area and peak area ratio (correlation coefficients 0.9992 and 0.9993, respectively). Repeatability of Analysis The day-to-day variability of analysis for sumatriptan content was established by conducting two separate sets of analyses on two separate occasions. </p><p>Similar results for each sample set were obtained on each occasion and these were in agreement with those achieved by HPLC (Table 2). Each analysis set comprised testing of on-going stability batches which had been stored at various conditions of heat and humidity. Table 2 Repeatability of results day-to-day Analysis set 1 - Sumatriptan content/mg cmP3 CE results HPLC Sample batch 1 Day 1 Day2 Mean Condition 1 11.9 11.9 11.9 11.8 Condition 2 11.8 11.6 11.7 11.7 Condition 3 11.7 11.7 11.7 11.7 Analysis set 2- Sumatriptan content/mg cmp3 CE results HPLC Sample batch 2 Day 1 Day 2 Mean Condition 1 11.9 11.6 11.8 11.6 Condition 2 11.7 11.6 11.7 11.7 Table 3 Sumatriptan content by CE and HPLC Sumatriptan content/mg cm-3 Sample Condition 1 (aliquot 1) Condition 1 (aliquot 2) Condition 2 (aliquot 1) Condition 2 (aliquot 2) Condition 1 (aliquot 1) Condition 1 (aliquot 2) Condition 2 (aliquot 1) Condition 2 (aliquot 2) Condition 1 (aliquot 1) Condition 1 (aliquot 2) Condition 2 (aliquot 1) Condition 2 (aliquot 2) Batch 2- Batch 3- Batch 4 - CE HPLC 11.5 11.6 11.6 11.6 11.6 11.7 11.6 11.7 11.7 11.8 11.8 11.8 11.6 11.7 11.6 11.7 11.7 11.8 11.8 11.8 11.7 11.7 11.6 11.7ANALYTICAL PROCEEDINGS, SEPTEMBER 1993, VOL 30 365 Repeatability of Separation In order to assess the ruggedness of the method the separation was performed on an alternative CE instrument (Beckman P/ACE 2000) using a capillary from a different supplier. The separation achieved showed an indentical migration order to that achieved on the earlier instrument and capillary. </p><p>Cross-correlation Between Sumatriptan Content Results by CE and HPLC Currently HPLC is employed for the determination of sumatriptan contents7 Sumatriptan content was determined by CE for three stability batches of sumatriptan injections (12 mg ~ m - ~ ) using external standardization. The comparison of the results obtained by CE and HPLC is shown in Table 3. Two aliquots were taken from each sample, and each aliquot was analysed in duplicate. The results are the mean of the two injections of each aliquot, good correlation was obtained. Conclusions A CE method has been employed for the determination of sumatriptan. The method gave good performance in terms of selectivity, precision, linearity and repeatability. </p><p>The good cross-correlation of CE and HPLC results suggests that CE could be employed for this and other quantitative analysis. References 1 Swartz, M. E., J. Liq. Chromatogr., 1991, 14, 923. 2 Altria, K. D., and Smith, N. W., J. Chromatogr., 1991,538,506. 3 Ackermans, M. T., Beckers, J. L., Everaerts, F. M., and Seelen, I. G. J. A., J. Chromatogr., 1992, 590, 341. 4 Dose, E. V., and Guiochon, G. A., Anal. Chern., 1991,63,1154. 5 Dawson, J . , Richards, D. A., Stable, R., Dixon, G. T., and Cockel. R., J. Clin. Hosp. Pharm., 1983, 8, 1. 6 McLaughlin, G. M., Nolan, J. A., Lindahl, J. L., Morrison, J. A., and Bronzert, T. J., J. Liq. Chromatogr., 1992, 15. 961. 7 Oxford, J., and Lant, M. S.. J. Chromatogr., 1989, 496, 137. Enantiomeric Separation of Ciprofibrate and Analogues Using a Cellulose-based Chiral Stationary Phase A. </p><p>Scott, P. R. Vojvodic and N. H. Anderson Sterling Winthrop Pharmaceuticals Research Division, Willowbum Avenue, Alnwick, Northumberland NE66 2JH B. J. Clark Pharmaceutical Chemistry, School of Pharmacy, University of Bradford, Bradford, West Yorkshire BD7 IDP It is known that individual enantiomers of chiral drugs can possess different potencies because of differences in pharmaco- dynamics, pharmacokinetic behaviour or metabolism. For new drug entities, unless the use of a racemate can be justified, major regulatory authorities require the development of a single enantiomer. The development of chiral stationary phases (CSPs) for gas-liquid chromatography (GLC) and high- performance liquid chromatography (HPLC) has greatly facilitated the determination of the enantiomeric purity of drugs. </p><p>Ciprofibrate {2-[4-(2,2-dichlorocyclopropyl)phenoxy]-2- methylpropanoic acid} (Fig. l), is a hypolipidaemic agent, currently marketed as a racemic mixture. For analytical purposes an enantioselective HPLC method was required.' A review of the literature on chiral separations revealed that cellulose triacetate had been successfully used to separate enantiomers which did not have a hydrogen bonding capability close to the chiral centre and therefore this phase was selected as a starting point for this programme.2 It was also proposed to gain an improved understanding of the chiral recognition sites of microcrystalline cellulose triacetate, by extending the examination to a number of the analogues of ciprofibrate. </p><p>Experimental Apparatus Chromatographic experiments were carried out using an HP 1090 Series 11 LC system and workstation (Hewlett-Packard, 0 Fort Collins, CO, USA). The column used was a Chiralcel CA- 1 (Daicel Corporation, Japan), 250 x 4.6 mm i.d. Conditions Column: Mobile phase: Temperature : Flow rate: Injection volume: Ultraviolet (UV) detection: Chiralcel CA-1, 250 X 4.6 mm i.d. 90 + 10 v/v ethanol-water. The mobile phase was filtered and de- gassed prior to use. 25 "C 0.5 cm3 min-' 10 mm3 of 1 mg cm-3 solution h 236 nm Results and Discussion Microcrystalline cellulose triacetate is prepared by the hetero- geneous acetylation of microcrystalline cellulose. As a CSP, it can have a broad applicability, it is compatible with reversed- phase solvents and it is capable of accepting a high analyte loading, which is useful in preparative and semi-preparative work. </p><p>The mobile phase and temperature parameters which were initially used were based on those found to give the successful separation of ciprofibrate methyl ester (b, Fig. 2). The chromatographic conditions used were 90 + 10 v/v ethanol- water at 0.5 cm3 min-', 25 "C, UV detection (A 236 nm) with a 10 mm3 injection volume. Ciprofibrate was derivatized to the methyl ester prior to chromatography, owing to the poor retention on the cellulose triacetate column of the parent compound. This is due to the presence of the carboxylic acid group so analogues having this function were also derivatized to the methyl ester. Analogues which Result from Variations in the C yclopropane Ring When both the chlorine atoms of ciprofibrate are replaced by either fluorine or bromine, baseline resolution of the enantio- Fig. </p><p>1 Structure of ciprofibrate366 0 ANALYTICAL PROCEEDINGS, SEPTEMBER 1993, VOL 30 torsion angle between the phenyl ring and cyclopropane ring for the ethyl compound h. a R’ = Br, R2 = H, R3 = Me, R4 = H b R’ = CI, R2 = H, R3 = Me, R4 = H c R’ = F, R2 = H, R3 = Me, R4 = H d R’ = Me, R2 = H, R3 = Et, R4 = H e R’ = CI, R2 = Me, R3 = Me, R4 = H f R’ = F, R2 = Me, R3 = Me, R4 = H g R’ = Br, R2 = Me, R3 = Me, R4 = H h R’ = CI, R2 = Et, R3 = Me, R4 = H i R’ = CI, R2 = H, R3 = Et, R4 = H j R’ = CI, R2 = H, R3 = Et, R4 = CI k R’ = F, R2 = H, R3 = Me, R4 = CI P I p R 5 = H q R5 = OMe r R5 = OCOMe s R5 = NH2 Fig. </p><p>2 Structures of ciprofibrate analogues Table 1 Resolution and selectivity values Sample a b d e f g h i k P Q r C .i S Retention time/ min PeakA Peak B 13.51 17.20 12.07 20.15 11.58 14.78 6.89 9.56 10.37 9.58 10.70 8.68 10.17 11.17 13.42 12.01 12.34 15.16 14.00 17.29 17.99 20.68 23.80 Rs 0.66 1.62 0.87 0.33 0.29 0.70 0.80 0.56 (r(A,B) k’(A) 1.39 2.38 2.00 2.02 1.43 1.89 0.72 1.14 1.39 1.39 1.68 1.17 1.16 1.54 2.35 2.02 1.33 2.09 1.33 2.50 3.50 1.19 4.17 k‘@) 3.30 4.04 2.70 1.59 1.79 2.79 3.32 4.95 mers was still achieved (Table 1). However, the dichloro analogue gave the best resolution. As regards retention, this increased according to Br>Cl>F (a, b, and c, Fig. 2). In the case of the ethyl ester analogue, d, in which methyl groups replace the chlorine atoms, poor resolution and retention were obtained. </p><p>This is considered to be due to an electronic and/or lipophilicity effect as the chlorine atom and methyl group are similar in size. However, when there is substitution of a methyl group for the hydrogen atom at the chiral centre, resolution of the dichloro analogue e was achieved but this was not the case with the fluoro and bromo analogues f and g, and this result was unexpected. Extension of this to an ethyl (h), rather than a methyl (e) group at the chiral centre, resulted in loss of resolution which is possibly due to the steric hindrance of the large sized ethyl group. This may result from the greater Comparison of Methyl and Ethyl Esters of Ciprofibrate Both the methyl and ethyl esters of ciprofibrate (b and i) were resolved on microcrystalline cellulose triacetate, with the methyl ester being more strongly retained. </p><p>This retention pattern is the opposite of that noted on a reversed-phase column and may be due to the possible polar character of the stationary phase. In addition, it was noted that compound j, a ring substituted ethyl ester, was not resolved. Phenyl Ring Substitution of Ciprofibrate Where the ciprofibrate phenyl ring is modified, such as with 3- chloro ring substitution k, enantiomeric resolution was not obtained although the analogues were retained on the column. Since the aromatic moiety is adjacent to the chiral centre, it would be expected to play a role in chiral recognition. The lack of resolution may be due to the steric and electronegative properties of the 3-chloro substituent. </p><p>Synthetic Precursors of Ciprofibrate Compound p is a relatively small, lipophilic molecule, and was well retained and well resolved. Compound q was also resolved, but compound r, an ester, was not; interestingly it was more strongly retained than the former analogues. It is possible that a dipolar interaction between the ester linkage and the cellulosic stationary phase prevents resolution of r. With the 1-amino precursor s , retention was greater than any other analogue tested and in addition it was partially resolved. This may be a result of a dipolar attraction between the negative nitrogen and the ester linkage of the stationary phase. This evidence of differences in behaviour between the acetate and the amino compounds indicates that a spatial relationship between the dipole of the group at the 1-position of the phenyl ring and the rest of the molecule can affect resolution of the enantiomers. </p><p>Conclusions The results illustrate that resolution on microcrystalline cellulose triacetate can be achieved with a range of modified structures from the base of the ciprofibrate model when carried out under constant chromatographic conditions. However, it is clear that certain chemical structure rules, for recognition, operate. It is suspected that no simple recognition process is occurring at a single site; rather that a number of differing chiral recognition sites are present on the microcrystalline cellulose triacetate, as has been previously reported by R i ~ z i ~ - ~ and Francotte .6,7 In order to give additonal corroboration of the analyte/stationary phase interactions, a further investi- gation is being completed to establish the dependence of retention and resolution of the enantiomers through the composition of the mobile phase. </p><p>References 1 Anderson, N. H., Johnston, D., and Vojvodic, P. R., J. Pharm. Biomed. Anal., 1992, 10, 501. 2 Wainer, I. W., A Practical Guide to the Selection and Use of HPLC Chiral Stationary Phases, J. T. Baker Chemical Co., Phillipsburg, NJ, USA, 1988. 3 Rizzi, A. M., J. Chromatogr., 1989. 478, 71. 4 Rizzi, A. M., J. Chromatogr., 1989, 478, 87. 5 Rizzi, A. M., J. Chromatogr., 1989, 478, 101. 6 Francotte, E., Wolf, R. M.., Lohmann, D., and Mueler, R., J . Chromatogr., 1985, 347, 25. </p><p>7 Francotte, E., and Wolf, R. M., Chirality, 1990, 2, 16.ANALYTICAL PROCEEDINGS, SEPTEMBER 1993, VOL 30 Inorganic Salts as Modifiers in the High-performance Liquid Chromatography Mobile Phase Used to Chromatograph Atenolol 367 Alison E. Bretnall and Thomas Cowen Bristol-Mvers Sauibb Pharmaceutical Research Institute, International Development Laboratories, Reeds Lane, Moreton, herseyside L46 70W Ionic counterions are used extensively in reversed-phase high- performance liquid chromatography (HPLC) to effect a chromatographic separation. They are often used to obtain a required specificity or to improve the efficiency of a particular separation. Some of the counterions most widely used to effect an ion-pair complex are the alkylsulfonic acids, e.g., heptansul- fonic acid. </p><p>In our experience, there are several disadvantages to using such ion-pair reagents, for instance long equilibration times, permanent modification of the chromatographic column and problems associated with mobile phase recycling. Also the chromatography can be poor with tailing peaks. These disadvantages may be overcome by using inorganic salts as modifiers in the mobile phase. An example of this is a recently developed procedure for the analysis of atenolol { (4-(2-hydroxy-3-isopropylaminopro- poxy)phenyl]acetamide} (Fig. 1), a synthetic beta-selective adrenoreceptor blocking agent, which is a relatively polar hydrophilic compound with basic character. Many of the published analytical methods'-' for atenolol utilize ion-pair reagents or alkylammonium salts. </p><p>In our experience, however, the chromatography with these methods is generally poor, with tailing peaks and short column life. In our laboratory atenolol has been chromatographed using a reversed-phase p-Bondapak phenyl column with a mobile phase containing potassium chloride as an inorganic modifier. This paper reviews the method, the effects of alternative columns and of varying the composition of the mobile phase. Atenolol Assay Method Chromatographic Parameters A 300 x 3.9 mm i.d. stainless steel column packed with p- Bondapak phenyl (Waters Associates, Millipore, Peter- borough) was used at 30 "C with a mobile phase containing methanol-1% ( d v ) aqueous potasssium chloride (1 + 3 v/v). The flow rate was 1.0 cm3 min-' and the detection by ultraviolet (UV) absorption at a wavelength of 240 nm. </p><p>Chromatography A solution of atenolol at a concentration of 1 mg cmP3 was prepared in methanol-water (1 + 1 v/v) and 20 mm3 were injected onto the HPLC system. Under these chromatographic conditions the atenolol eluted at 5.2 min as a sharp peak with a N I H Me Fig. 1 Structure of atenolol tailing factor of 1.6. The method was successfully validated for precision, accuracy, specificity and linearity of response. Atenolol possesses a number of known impurities and degradants which are possibly present in the bulk drug material and may therefore be found in the finished product. Of these six known compounds can be resolved from atenolol under the conditions of the assay (Table 1). The specificity of this method was only validated using 1% ( d v ) aqueous potassium chloride in the mobile phase. </p><p>Variations in Mobile Phase Composition and Column As inorganic salts have been used previously for methods in our laboratories, an investigation into the effects of varying the mobile phase composition and the reversed-phase HPLC column was undertaken. Variation of the Concentration of Potassium Chloride in the Mobile Phase A range of mobile phases containing 0.01, 0.1,0.5, 1.0,2.0 and 4.0% ( d v ) aqueous potassium chloride-methanol(1 + 3 v/v) were prepared and with the chromatographic parameters described previously, used to chromatograph the atenolol solution (Fig. 2). The retention time of the atenolol peak was consistent at 5.5 min for 0.5-4.0% (m/v) aqueous potassium chloride; however, the tailing factor decreased from 2.1 to 1.2, respectively. </p><p>For the lower concentrations of 0.1 and 0.01% ( d v ) aqueous potassium chloride, the retention time increased from 7 to 15 min and the peak tailing increased. (Tailing factor greater than 3.0 for both concentrations.) Effect of the Cationic Chloride Salt in the Aqueous Component of the Mobile Phase A range of mobile phases were prepared containing 1% ( d v ) aqueous chloride salt-methanol (1 + 3 v/v), where the cation was zinc, potassium, rubidium, sodium, magnesium or calcium. Atenolol was chromatographed using the above range of mobile phases (Fig. 3) and the conditions described in the chromatographic parameters. The retention time of the atenolol peak was consistent at 5.5 min whereas the tailing factor varied. </p><p>The mobile phases containing zinc, potassium and rubidium all gave peaks with tailing factors of less than 2.0. Those containing sodium, magnesium and calcium gave peaks with tailing factors greater than 2.0. Table 1 Retention times of atenolol and six known impurities Retention time/min Atenolol 5.2 (4-Hydroxypheny1)acetamide 4.6 Bis-[3-(4'-carbamoylmet hylphenoxy)-2-hydroxy- prop yllisoprop ylamine 16.0 Fumaric acid 2.7 4-(2-Hydroxy-3-isopropylaminopropoxy)- phenylacectic acid 6.1 4-( 2-Hydroxy-3-ch1oropropoxy)phenylace tamide 13.2 [4-(2,3-Epoxypropoxy)phenyl]acetamide 10.8368 ANALYTICAL PROCEEDINGS, SEPTEMBER 1993, VOL 30 300 > E % 200 2! 8 100 0" 1 0 P +- al +- 0 I I 1 I I I I I I 1 1 1 2 4 6 8 10 12 14 16 18 20 22 24 E I ut i on ti m elm in Fig. </p><p>2 Chromatogram of atenolol (1 mg cmP3) using mobile phase containing A , 4.0; B, 2.0; C, 1.0; D, 0.5; E, 0.1; and F, 0.01% m/v aqueous potassium chloride-methanol (1 + 3 v/v) 800 700 $ 600 500 2 400 300 1 Q fn L 0 +- 0" 200 100 0 1 2 3 4 5 6 7 8 9 Elution time/min Fig. 3 Chromatogram of atenolol (1 mg ~ r n - ~ ) using mobile phase containing 1% m/v aqueous chloride salt-methanol (1 + 3 v/v) where the chloride salt was: A, ZnC1,; B, KCI; C, RbCl; D, NaCl; E, MgCl,; and F, CaCI2 There was also an increase in the intensity of the UV response for the atenolol with zinc and calcium. However, the use of calcium is not recommended as a rapid deterioration of the chromatographic performance was observed. Effect of the Anionic Potassium Salt in the Aqueous Component of the Mobile Phase A range of mobile phases were prepared containing 1% ( d v ) concentration aqueous potassium bromide, potassium phos- phate and potassium sulfate solution, respectively, in methanol (1 + 3 v/v). </p><p>Each was used to chromatograph the atenolol solution (1 mg cmW3) using the chromatographic parameters described previously. The retention time for the atenolol peak was between 4.2 and 5.2 min and the tailing factor was 1.5. Effect of the Organic Modifier A mobile phase containing 1% ( d v ) aqueous potassium chloride-acetonitrile (1 + 3 v/v) was used to chromatograph the atenolol solution (1 mg ~ m - ~ ) . On comparison with the mobile phase containing methanol, a significant loss in chroma- tography was observed. The retention time was reduced to 3.5 min and the peak showed fronting and tailing. </p><p>Alternative HPLC Columns A mobile phase was prepared containing 1% ( d v ) aqueous potassium chloride-methanol (1 + 3 v/v) and three columns Table 2 Retention times and peak shapes of atenolol on alternative HPLC columns Retention Column t ime/mi n Peak shape p-Bondapak CIS 4.5 Sharp peak, tailing factor 1.7 300 x 3.9 mm i.d. (Waters) Hypersil BDS CI8 5 pm 150 x 4.6 mm i.d. (Shandon) 8 x 100mm (Waters) 2.5 Sharp peak but with significant tailing Silica radial pack 10 pm 10.5 Significant fronting and tailingANALYTICAL PROCEEDINGS, SEPTEMBER 1993, VOL 30 369 were used to chromatograph the atenolol solution (Table 2, Fig. 4). A p-Bondapak CIS column gave comparable chroma- tography to that obtained with the p-Bondapak phenyl used previously, whereas a Hypersil base-deactivated silica column which is claimed to be specially designed for the chroma- tography of basic compounds, elutes a peak with significant '0° 1 b 300 n loo 0 k 2 1 2 3 4 B L I I 1 I I 1 I I 1 1 5 6 7 8 9 10 11 12 13 14 15 E I utio n ti m e/m i n Fig. </p><p>4 Chromatogram of atenolol (1 mg using columns, A, Hypersil BDS CI8; B, pBondapak CIS; and C, Silica radial pak with mobile phase containing 1% m/v aqueous potassium chloride- methanol (1 + 3 v/v) tailing. (It should be noted that without the inorganic modifier the atenolol showed gross peak distortion.) Chromatography was also performed on a silica packed column and gave significantly greater retention of atenolol with increased tailing. Conclusions Atenolol has been chromatographed successfully using a reversed-phase HPLC system utilizing potassium chloride as inorganic modifier, and fully validated as a stability indicating assay. </p><p>Satisfactory chromatography has also been obtained €or atenolol using various inorganic salts as mobile phase modifiers which may be applicable to other basic compounds. The enhanced UV response for atenolol when chromato- graphed using zinc and calcium ions was considered of interest and will be investigated in the future. References Owino, E., Clark, B. J., and Fell, A. F., J . Chromatogr. Sci., 1991, 29, 4.50. Yee, Y., Rubin, R., and Blaschke, T. F., J . Chrornatogr., 1979, 171, 3.57. Miller, R. B.. J. Pharm. Biomed. Anal., 1991, 9, 849. Pawlak, Z., and Clark, B. J., J . </p><p>Pharm. Biomed. Anal., 1992,10, 329. British Pharmacopoeia 1988, HM Stationery Office, London, 1988, vol. 1, p. 49 and vol. 2, p. 903. Determination of Dextromethorphan and its Metabolites in Human Urine Using Solid-phase Extraction and Reversed-phase High-performance Liquid Chromatography Rachel Howling SmithKline Beecham Consumer Brands, St. Georges Avenue, Weybridge, Surrey KT13 ODE George Hutchinson Anachem, Charles Street, Luton, Bedfordshire LU2 OEB Dextromethorphan (Dm) is an antitussive widely used in the over-the-counter (OTC) cough-cold market. In humans it is metabolized to dextrorphan (Dt), 3-hydroxymorphinan (3- OH) and 3-methoxymorphinan (3-Me0). All three of these metabolites form conjugates with glucuronic acid at the 3- hydroxy group position. </p><p>This metabolism is under genetic control, co-segregating with the debrisoquine polymorphism.' Three different metabolizers can be identified, Group 1 (extensive metabolizers), Group 2 and Group 3 (poor metab- olizers).* Poor metabolizers have a Dt/Dm ratio below 1 in their urine collected for 8 h after an oral dose of Dm and make up 5-10% of the Caucasian population, whereas the group 2 metabolizers have a Dt/Dm ratio between 1 and 10, and the extensive metabolizers have a ratio above ten. Various analytical methods are available to determine Dm in human urine, including: thin-layer chromatography (TLC);3 gas chromatography (GC);2 high-performance liquid chroma- tography (HPLC) with ultraviolet (UV) d e t e ~ t i o n ; ~ and HPLC with fluorescence d e t e ~ t i o n . </p><p>~ All these methods are either only comparative in the case of TLC or require time consuming sample preparation. There are also two methods published which use solid-phase extraction but one requires the extract from the cartrid e to be dried down, thus reducing the ease of automation: and the other only supports phenotype determinati~n.~ Experimental One hundred cubic millimetres of urine are pipetted into a 4 cm3 glass sample tube, to which 100 mm3 of a 55 pg cm-3 codeine solution (IS) and 1 cm3 of 2 mg cm-3 P-glucuronidase in 0.5 mol dm-3 sodium acetate (pH 5 ) solution are added. The mixture is incubated at 37 "C overni ht. cartridges are preconditioned with 1000 mm3 of acetonitrile and 1000 mm3 of water, followed by 50 mm3 of air. The 1200 mm3 of incubated urine sample and buffer are added followed by 1000 mm3 of air. </p><p>The cartridge is then washed using 1000 mm3 of water and 1000 mm3 of acetonitrile, again both followed by 1000 mm3 air. The Dm and its metabolites are eluted with 1000 mm3 of 60% acetonitrile-40% 0.02 mol dm-3 NaH2P04 pH 3.6 and 1000 mm3 air into 3.5 cm3 polyethylene collection tubes. After mixing by bubbling through air, 5 mm3 of the eluted solution are injected on to the HPLC. All the above procedure is carried out by the ASPEC without any operator intervention. Chromatographic separation is achieved isocratically, on a 15 cm x 2.2 mm i.d. Spherisorb 5 pm CN analytical column fitted with a 5SCN guard column using a 45% acetonitrile-55% 0.01 mol dm-3 NaH2P04, containing 75 mm3 of orthophos- phoric acid per dm3, the mobile phase being run at Using a Gilson ASPEC 100 mg, 1 cm B Phenyl Bond Elute370 il ANALYTICAL PROCEEDINGS, SEPTEMBER 1993, VOL 30 1 OO( > E $ . </p><p>0 al a 0 D I 10 Time/min 600 > E % . 0 Q) a a , ( b ) A I 1 0 10 Ti me/m i n Fig. 1 Typical chromatograms for ( a ) spiked and ( b ) blank human urine extracts. A, Codeine; B, 3-OH; C, Dt; D, 3-Me0; E, Dm 0.25 cm3 min-'. Fluorescence detection is used with an excitation wavelength of 230 nm and and emission wavelength of 312 nm. This gives the following retention times: 5.0 min codeine, 6.5 min 3-OH, 7.0 rnin Dt, 8.0 rnin 3-Meo and 9.0 min for Dm. Typical chromatograms for blank and spiked human urine extracts are as shown in Fig. 1. Calibration is camed out using six spiked urine samples and a curve obtained using unweighted linear regression. </p><p>Results For the purpose of validation, 1000 mm3 of 0.5 mol dm-3 sodium acetate buffer, containing 2 mg cm-3 P-glucuronidase, are added to all samples (to simulate the in-use conditions). If the 100 mm3 of codeine, IS, is not used then it is replaced with distilled water. The limit of quantification is taken as 0.5 pg cmV3 for all four species and this is the lowest level which gave suitable accuracy and precision results. Extraction recoveries are 100% for all four species in the range 0.5-10.0 pg ~ m - ~ . Calibration graphs show correlation coefficient values, Y, >0.99 for Dt, 3-Me0 and 3-OH and >0.98 for Dm for the range of 0.5-10 pg cmV3. Within batch precision gave relative standard deviations (RSDs) of </p> <p>Urine samples obtained from volunteers dosed with Dm stored at -20 "C for four weeks showed no signs of degradation. Conclusions This paper describes an accurate, precise and robust assay for the determination of Dm and its metabolites in human urine which is suitable for use in clinical studies and for the determination of p450 phenotype status. References 1 Schmid, B., Bircher, J., Preisig, R., and Kupfer, A., Clin. Pharmacol. Ther., 1985,38, 618. 2 Pfaff, G., Breigel, P., and Lamprecht, I., Znt. J. Pharm., 1983, 14, 173. 3 Guttendorf, R. J., Britto, M., Blouin, R. A., Foster, T. </p><p>S., John, W., Pittman, K. A., and Wedlund, P. J., Br. J. Clin. Pharmacol., 1990, 29, 373. 4 Hilderbrand, M., Seifert, W., and Reichenberger, A., Eur. J. Clin. Pharmacol., 1989,36, 315. 5 Chen, Z. R., Somagyi, A. A., and Bochner, R., Ther. Drug. Monit., 1990, 12, 97. 6 Wenk, M., Todesco, L., Keller, B., and Follath, F., J. Pharm. Biomed. Anal., 1991, 9, 342. 7 Jacqz-Aigrain, E., Menard, Y., Popan, M., and Mathieu, H., J. Chromatogr., 1989, 495, 361.ANALYTICAL PROCEEDINGS, SEPTEMBER 1993, VOL 30 0 1 - 2 - 3 - C .- E F 5 \ - E" 10 15 20 371 CS = 0.5 Solvent front =C -cs = 0.1 ': - - R Development of a Method for the Detection of Angiotensin Converting Enzyme Inhibitors Using Electron Capture-Gas Chromatography Detection K. M. Sereda, T. C. Hardman, M. R. Dilloway and A. </p><p>F. Lant Department of Clinical Pharrnacolog y and Therapeutics, Chelsea and Westminster Hospital, London SWIO 9NH Angiotensin converting enzyme (ACE) inhibitors are used in the treatment of essential hypertension and heart failure. The majority of the therapeutic compounds in this group are prodrugs, requiring activation after oral administration to exert their effect. The ACE inhibitors investigated included the active drug captopril, the prodrugs benazepril, ramipril, enalapril and perindopril, as well as the primary active metabolites of these prodrugs benazeprilat , ramiprilat , enala- prilat and perindoprilat . De-esterification of the prodrugs results in the dicarboxylic acid metabolites which, in common with the other members of the class, are extremely water soluble. </p><p>ACE inhibitors were designed to mimic natural peptides and as such they are effective in relatively small doses (1-10 mg); as a result, analytical methods currently employed to detect individual compounds are varied and complex ranging from radioimmunoassay ' to gas chromatography-mass spec- trometry (GC-MS).2 It has been shown that good gas chromatographic (GC) properties can be conferred upon members of this class of dru s by derivatizing with pentafluorobenzyl bromide ( P F B - B I - ) . ~ ~ The pentafluorobenzyl group is an excellent electrophore and results in a high electron-capture detector (ECD) response to these compounds. However, the use of PFB-Br as a deriva- tizing agent is hampered by the need to add an excess of PFB- Br. The excess of reagent tends to overload the EC detector if it is not removed from the sample prior to its injection onto the column. </p><p>Different methods of removing the excess of PFB-Br after derivatization which have been suggested include evapo- ration, extraction into an aqueous phase, separation on a silica gel column, coupling the excess of reagent to an aminophenol to give a product that can be extracted with water or by solvent venting systems attached to the GC.s This paper describes the determination of ACE inhibitors and their active metabolites by derivatization with PFB-Br and electron-capture detection, employing a temperature- controlled evaporation procedure to remove the excess of derivatizing agent. Experimental The ACE inhibitor ramiprilat was used as an internal standard for the calibration of the response of the system to the other compounds, the drug perindopril being used when the system's response to ramiprilat was investigated. </p><p>Pure stock standard solutions of the internal standard, drugs and metabolites were made up to a concentration of 1 mg cm-3 in methanol and stored below 4 "C. The derivatization procedure included the addition of potassium acetate (10 mg), acetone (3 cm3), internal standard (ramiprilat, 100 pg, 100 mm3) and an aliquot (1-100 mm3) of the stock solution of the ACE inhibitor, or metabolite, being investigated to a 10 cm3 vial. PFB-Br (100 mm3) was then added, the vial capped and the mixture vortexed at 70 "C for 2 h. After derivatization the sample was left to cool. An aliquot (250 mm3 of the cooled solution was evaporated to dryness in a 0.5 cm vial at 100 "C for 15 min. </p><p>Following evaporation, the Sam le was taken up in acetone (250 mm3) and injected (0.5 mm ) onto the gas chromato raph. A Varian 3400 GC equipped with a nickel (6 Ni) radioactive ECD unit and coiled glass tube column (30 m x 0.252 mm i.d.) packed with a methylsilicone liquid phase was used in the experiment. The column, injector and detector port tempera- tures were set to 295,300 and 310 "C, respectively. The carrier gas, helium, pressure was 25 psi and the argon-methane flow was set to 30 cm3 min-'. 2 s B Results and Discussion Standard curves were obtained for all nine componds investi- gated with the correlation coefficient (Y) being obtained for each. All the nine compounds investigated had different retention times, achieving good separation between each peak,372 ANALYTICAL PROCEEDINGS, SEPTEMBER 1993, VOL 30 Table 1 Retention times (RT), relative standard deviations (RSD) and limits of detection (LOD) of the ACE inhibitors and active metabolites investigated LOD (pg on- Drug RT/min RSD (%) column) Captopril Perindopril Enalapril Perindoprilat Ramipril Enalaprilat Benazepril Ramiprilat Benazeprilat 3.69 4.69 7.15 8.54 10.33 12.59 13.96 18.78 25.80 4.80 2.93 3.79 5.55 2.00 2.81 6.81 4.08 2.18 1.35 0.87 1.03 1.55 0.56 0.83 1.89 0.79 0.59 hence, if necessary, permitting the simultaneous measurement of all the ACE inhibitors investigated here. An example of a typical chromatogram is shown in Fig. </p><p>1. The standard curves were all based on 60 readings and the r values obtained were always greater than 0.998. </p><p>The retention times (RT) of the ACE inhibitors investigated, together with their relative standard deviations (RSD) and limits of detection (LOD), are given in Table 1. The relative standard deviations indicated the precision of the injections and the limits of detection were a measure of the smallest amount of drug which can be detected using this method. The work presented here tested whether electron capture-gas chromatography (EC-GC) could be used as an analytical tool for the detection of ACE inhibitors. For EC-GC to be used in the analysis of this group of chemicals, two problems had to be overcome. Firstly, as mono- and di- acid moieties these compounds had to be converted into a form that could be volatilized in the gas chromatograph, and secondly, the molecule had to be altered in order to make it detectable by electron capture, since ACE inhibitors possess little electron capture ability. </p><p>Pentafluorobenzyl bromide (PFB-Br) was chosen as the molecule to be attached to the parent compound to enable volatilization and detection as it was known to be a successful derivatizing agent of carboxylic acid groups and had been previously used in the analysis of c a p t ~ p r i l . ~ Potassium acetate used as the catalyst has been reported to impose a selectivity upon the PFB-Br for the alkylation of carboxylic acid groups only. Ramiprilat was chosen as the internal standard, as it stood alone on the chromatograph with no interfering peaks within 5 min either side. </p><p>In the present study, evaporation of the derivatized samples at 100 "C for 15 min proved to be an effective way of eliminating, through volatilization, the excess of PFB-Br reagent and its derivatives of low molecular mass. This procedure resulted in a clearer chromatogram with fully resolved peaks at earlier times in the elution, with the added benefit that the detector, through not being overloaded, easily remained within its linear range. This modification solved many problems such as detector desensitization and hence the procedure gave reliable and reproducible results. Using ramiprilat as the internal standard and the clean up procedure described above, the assay was applied to all the drugs. The results presented in this paper showed that for each of the compounds, the routine analysis gave linear standard curves over the range 0.31-15.75 ng of drug going on the column. </p><p>Work to establish day-to-day variation showed the deriva- tized ACE inhibitors investigated to be stable for at least 1 month under normal laboratory conditions. This was achieved by reinjecting a solution daily over the period of study and comparing the relative heights. This work has shown that a traditional method of detection such as electron capture remains a useful tool in pharma- ceutical research despite the increasing use of more expensive and elaborate techniques such as GC-MS. References 1 Biollaz, J., Schelling, J . L., Jacot Des Combes, B . , Brunner, D . B . , Desponds, G., Brunner, H. R . , Ulm, E. H., Hichens, M., and Gomez, H. J., Br. </p><p>J. Clin. Pharmacol., 1982, 14, 363. 2 Kaiser, G., Ackermann, R., Dieterle, W., and Dubois, J . , J. Chromatogr., Biomed. Appl. 1987, 419, 123. 3 Ito, T., Matsuki, Y., Kurihara, H . , and Nambara, T., J . Chromatogr., 1987, 417, 79. 4 Dilloway, M. R . , and Hardman, T. C., Proceedings of the Fourth Annual Conference on Ace Inhibitors, London, 1991. 5 Gyllenhaal, O., Brotell, H . , and Sandgren, B., J. Chromatogr. 1976, 122, 471. Determination of Total and Ionic Chloride and Bromide in a Cross- linked Quaternary Ammonium-substituted Polymethacrylate by Ion Chromatography Ian D. Smith, Paul D. Blackler and David G. Waters Analytical Sciences Department, Smith Kline Beecham Pharmaceuticals, Old Powder Mills, Leigh, Kent TNII 9AN A quaternized polymethacrylate resin, SK&F97426-A, is undergoing development as a potential bile acid sequestrant. Bile acid sequestrants are commonly ion-exchange resins that bind bile acids in the gut and cause them to be excreted in the faeces. </p><p>They are used to treat hypercholesterolaemia where dietary control of lipids has proved inadequate. This paper discusses methods employed for the determi- nation of ionic and total halides in SK&F97426-A. Chloride and bromide levels were determined as a means of measuring batch-to-batch variation in the final product. Ionic bromide is toxic and has to be controlled to low levels. The specification for SK&F97426-A requires a maximum of 0.2% m/m bromide in approximately 10% m/m chloride. The halides were originally determined potentiometrically , ionic halides after displacement by nitrate and total halides after oxygen flask combustion. Potentiometry had a limit of detection of approximately 0.5% m/m for bromide in SK&F97426-A. </p><p>Once the lower specification was set, a more sensitive method had to be found and ion chromatography was adopted. The concentration of nitrate used as the displacer was too high to allow a satisfactory separation of the nitrate and bromide peaks. Nitrate was replaced with sulfate, which is wellANALYTICAL PROCEEDINGS, SEPTEMBER 1993, VOL 30 373 50 > E . fn 0 a fn 2 L c 0 al c 2 I 1; 0 0.8 1.6 2.4 3.2 4.0 4.8 5.6 6.4 7.2 8.0 a Retention t i m e h i n Fig. 1 Chromatogram of bromide standard solution showing bromide peak and sulfate from carry-over separated from both the analytes of interest and quantitatively displaced both anions from the polymeric matrix. Experimental Instrumental The ion chromatograph was a Dionex Model 2010i with an anion micromembrane suppressor and a conductivity detector. </p><p>A Dionex 25 cm IonPac AS4A analytical column with a 5 cm IonPac AG4A guard column were used at ambient tempera- ture. Sample injection was performed with a Perkin-Elmer ISS-100 autosampler with a 20 mm3 stainless steel injection loop, operated in filled-loop mode. The mobile phase was 0.75 mmol dm-3 sodium hydrogencarbonate and 2.2 mmol dm-3 sodium carbonate at a flow rate of 2.0 cm3 min-l. The suppressor regenerant solution was 25 mmol dm-3 sulfuric acid at a flow rate of 5 cm3 min-l. A Waters 860 ‘ExpertEase’ chromatography data system was used. </p><p>High intensity light sources housed in an enclosed safety cabinet were used to initiate the combustion in the flask. The combustion chamber, flasks and sample holders were prepared by an in-house technical service department. Sample Preparation As SK&F97426-A is hygroscopic, samples were dried under vacuum at 60 “C overnight before analysis and sample weigh- ings performed as rapidly as possible to reduce the risk of the sample adsorbing moisture. To avoid contamination during sample preparation, all apparatus was repeatedly rinsed with high-performance liquid chromatography (HPLC) grade water before use. Disposable latex gloves were worn for all sample handling and preparation and all reagents used were of the highest purity. </p><p>Ionic chloride und bromide The exchange of sulfate for the analyte ions was performed in plastic beakers as beads of the sample tended to adhere to glass. A small amount of acetone was added as a wetting agent to disperse the polymer. Approximately 400 mg of SK&F97426-A were stirred with 100 cm3 of 20% m/v sodium sulfate solution for an hour. The slurry was filtered, the filter cake washed and the filtrate and washings pooled and made up to 250 cm3 with HPLC-grade water. Separate dilutions were prepared for ionic bromide (1 + 19) and ionic chloride (1 + 249), which were analysed against 0.5 pg cm-3 bromide and 1.5 pg cmW3 chloride standard solutions, respectively. Total chloride and bromide Combustion flasks containing 50 cm3 of 10 mmol dm-3 sodium hydroxide solution were purged with oxygen. </p><p>Approximately 50 mg of SK&F97426-A were weighed into gelatine capsules, which were wrapped in a filter paper thimble and placed in a platinum mesh sample holder. After combustion, the flask was shaken vigorously by hand for 5 min to dissolve the combustion products. The contents and washings of the flask were made up to 250 cm3 with HPLC-grade water. This solution was filtered to 0.45 pm and analysed directly for total bromide against a 0.5 pg cm-3 standard solution and diluted 1 + 19 to determine total chloride against a 1.5 pg cm-3 standard solution. Chromatography The bromide peak eluted earlier in samples for ionic halides than in the standards. This was ascribed to a displacement effect of sulfate moving through the column. </p><p>Chloride eluted at approximately 1.4 min, bromide at approximately 2.2 min and sulfate at approximately 4.9 min. Fig. 1 shows a chromatogram of a bromide standard solution and Fig. 2 of an ionic chloride sample solution. There is some carry-over of sulfate visible in Fig. 1, thought to be due to the use of a stainless steel injector loop. Method Validation Linearity of response Linearity of response was tested for both analytes over relatively narrow concentration ranges, 0.5-3 pg cm-3 for chloride (approximately 5-28% m/m) and 0.1-1.5 pg cm-3 for bromide (approximately 0.02-0.36% d m ) . Satisfactory linearity for each of the analytes was demonstrated over the concentration range examined (220.998). Negligible inter- cepts suggested that single-point standards would not cause significant errors in calculated concentrations. Precision of replicate injections Ten injections were made of standard solutions of each analyte and the precision was measured by the YO relative standard deviation (%RSD) for the peak areas. It was found to be 1.0% for chloride and 5.1% for bromide. Precision of replicate preparations Replicate injections were made of each of several separately- prepared sample solutions. The results were subjected to one- way analysis of variance (ANOVA). Acceptable precision for374 ANALYTICAL PROCEEDINGS, SEPTEMBER 1993, VOL 30 180 0 I I I I 1 I I I I 1 0.8 1.6 2.4 3.2 4.0 4.8 5.6 6.4 7.2 8.0 Retention ti me/m i n Fig. 2 Chromatogram of ionic chloride sample solutions showing chloride and sulfate peaks replicate sample preparations was found for both total and ionic chloride and bromide. Standard and sample stability Standard solutions were stored in stoppered, clear glass calibrated flasks at room temperature and analysed against freshly-prepared standard solutions. Complete stability over 7 d was demonstrated. Sample solutions could be left in contact with the polymer overnight and sample solutions could be analysed up to a day after separation from the polymer without significant effects on the results. Ruggedness Assay results at flow rates of 1.8 and 2.2 cm3 min-' were compared with those at 2.0 cm3 min-l. Assays with mobile phases of 90 and 110% of the nominal concentrations of carbonate and hydrogencarbonate were compared with the nominal concentrations. The method was shown to be robust with respect to the mobile phase flow rate and composition over the ranges examined. Limits of detection The limit of detection was defined as the concentration that would give a peak height equal to three times the baseline noise. For chloride it was found to be 0.125% m/m and for bromide 0.008% m/m. Accuracy To test the methods, SK&F97426-A was analysed for total chloride and bromide by neutron activation analysis. This indicated that our methods were of acceptable accuracy. Conclusions Methods for the determination of ionic and total halides in SK&F97426-A and validation work undertaken with these methods were discussed. These standard methods have been in regular use for some time in our laboratory.</p></section></art-body></article></istex:document></istex:metadataXml><mods version="3.6"><titleInfo><title>Short papers in pharmaceutical analysis</title></titleInfo><titleInfo type="alternative" contentType="CDATA"><title>Short papers in pharmaceutical analysis</title></titleInfo><name type="personal"><namePart type="given">Chris J.</namePart><namePart type="family">Goodwin</namePart></name><name type="personal"><namePart type="given">K. D.</namePart><namePart type="family">Altria</namePart></name><name type="personal"><namePart type="given">S. D.</namePart><namePart type="family">Filbey</namePart></name><name type="personal"><namePart type="given">A.</namePart><namePart type="family">Scott</namePart></name><name type="personal"><namePart type="given">P. R.</namePart><namePart type="family">Vojvodic</namePart></name><name type="personal"><namePart type="given">N. H.</namePart><namePart type="family">Anderson</namePart></name><name type="personal"><namePart type="given">B. J.</namePart><namePart type="family">Clark</namePart></name><name type="personal"><namePart type="given">Alison E.</namePart><namePart type="family">Bretnall</namePart></name><name type="personal"><namePart type="given">Thomas</namePart><namePart type="family">Cowen</namePart></name><name type="personal"><namePart type="given">Rachel</namePart><namePart type="family">Howling</namePart></name><name type="personal"><namePart type="given">George</namePart><namePart type="family">Hutchinson</namePart></name><name type="personal"><namePart type="given">K. M.</namePart><namePart type="family">Sereda</namePart></name><name type="personal"><namePart type="given">T. C.</namePart><namePart type="family">Hardman</namePart></name><name type="personal"><namePart type="given">M. R.</namePart><namePart type="family">Dilloway</namePart></name><name type="personal"><namePart type="given">A. F.</namePart><namePart type="family">Lant</namePart></name><name type="personal"><namePart type="given">Ian D.</namePart><namePart type="family">Smith</namePart></name><name type="personal"><namePart type="given">Paul D.</namePart><namePart type="family">Blackler</namePart></name><name type="personal"><namePart type="given">David G.</namePart><namePart type="family">Waters</namePart></name><typeOfResource>text</typeOfResource><genre type="article" displayLabel="ART" authority="ISTEX" authorityURI="https://content-type.data.istex.fr" valueURI="https://content-type.data.istex.fr/ark:/67375/XTP-6N5SZHKN-D"></genre><originInfo><publisher>The Royal Society of Chemistry.</publisher><dateIssued encoding="w3cdtf">1993</dateIssued><copyrightDate encoding="w3cdtf">1993</copyrightDate></originInfo><language><languageTerm type="code" authority="iso639-2b">eng</languageTerm><languageTerm type="code" authority="rfc3066">en</languageTerm></language><relatedItem type="host"><titleInfo><title>Analytical Proceedings</title></titleInfo><titleInfo type="abbreviated"><title>Anal. Proc.</title></titleInfo><genre type="journal" authority="ISTEX" authorityURI="https://publication-type.data.istex.fr" valueURI="https://publication-type.data.istex.fr/ark:/67375/JMC-0GLKJH51-B">journal</genre><originInfo><publisher>The Royal Society of Chemistry.</publisher><dateIssued encoding="w3cdtf">1993</dateIssued></originInfo><identifier type="ISSN">0144-557X</identifier><identifier type="coden">ANPRDI</identifier><identifier type="RSC-sercode">AP</identifier><part><date>1993</date><detail type="issue"><caption>no.</caption><number>9</number></detail><extent unit="pages"><start>361</start><end>374</end><total>14</total></extent></part></relatedItem><identifier type="istex">87BC31C4076810D9EF32DC83ECCADA8A4FFEF340</identifier><identifier type="ark">ark:/67375/QHD-JMTF2065-V</identifier><identifier type="DOI">10.1039/AP9933000361</identifier><identifier type="ms-id">AP9933000361</identifier><accessCondition type="use and reproduction" contentType="copyright">This journal is © The Royal Society of Chemistry</accessCondition><recordInfo><recordContentSource authority="ISTEX" authorityURI="https://loaded-corpus.data.istex.fr" valueURI="https://loaded-corpus.data.istex.fr/ark:/67375/XBH-Z6ZBG4DQ-N">rsc-journals</recordContentSource><recordOrigin>The Royal Society of Chemistry</recordOrigin></recordInfo></mods><json:item><extension>json</extension><original>false</original><mimetype>application/json</mimetype><uri>https://api.istex.fr/ark:/67375/QHD-JMTF2065-V/record.json</uri></json:item></metadata><annexes><json:item><extension>gif</extension><original>true</original><mimetype>image/gif</mimetype><uri>https://api.istex.fr/ark:/67375/QHD-JMTF2065-V/annexes.gif</uri></json:item></annexes><serie></serie></istex></record>Pour manipuler ce document sous Unix (Dilib)
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