Uncertainty Related to the Use of

Transcription

1 Uncetainty Related to the Use of Relative Retention Times in Phamaceutical Analysis Piete Dehouck, a Doa Visky, a,b Zsuzsanna Kovács, a,b Béla Noszál, b Ewin Adams a and Jos Hoogmatens a a Laboatoy fo Phamaceutical Chemisty and Dug Analysis, Leuven, Belgium, b Depatment of Phamaceutical Chemisty, Semmelweis Univesity, Budapest, Hungay. One way to identify peaks in a chomatogam is by thei elative etention time (RRT). Howeve, because of a lack of hamonization in the calculation of RRT, intepetation of esults may be difficult. In both the Euopean Phamacopoeia (Ph. Eu.) and the United States Phamacopeia, much confusion emains because no method fo the detemination of hold-up times is pescibed. Also, the influence of the stationay phase must be taken into account. In this study, five sepaations pescibed by the Ph. Eu. wee pefomed on 9 diffeent evesed-phase liquid chomatogaphy C8 columns and the vaiability of RRTs between them was studied. It is shown that the RRT alone is not sufficient fo coect peak identification. Intoduction Peaks coesponding to specified impuities must be identified in a chomatogam. One way to achieve this is though the elative etention time (RRT). The RRT can be defined as the etention elative to a standad, o (t R t M ) / (t R(st) t M ) [] whee t R is the etention time of the component of inteest, t R(st) the etention time of the standad and t M the hold-up time. The main peak in the chomatogam is usually consideed to be the standad ( efeence peak). The hold-up time t M can be defined as the time it takes fo a small, unetained compound that completely pemeates the stationay-phase poes, to be eluted fom the chomatogaphic column. In fact, the atio used fo the calculation of is simila to the atio of mass distibution atios. The United States Phamacopeia (USP) has used Equation fo some time. Howeve, is not always calculated this way. Until the Euopean Phamacopoeia (Ph. Eu.) defined as the atio of the two etention times of the consideed peaks, o a/b = t,b / t,a [] with t,b the etention time of the peak of inteest and t,a the etention time of the efeence peak. Fom, t M has been taken into account and a fomula equal to Equation is descibed. Unfotunately, RRT values in monogaphs wee not adapted accodingly and it is not always cetain that the new Equation has been applied in monogaphs published since. This lack of hamonization aound the calculation of RRT has led to much confusion among analysts. Many texts still descibe as a atio of two etention times without consideing the subtaction of t M. Howeve, even when Equation is applied, confusion emains ove how to measue t M. Diffeent LC GC Euope Novembe

2 compounds, such as thiouea, uacil, nicotinic acid and nitate, have been cited in the liteatue as makes of t M. Nevetheless, no hamonized pocedue fo its measuement can be found and even pominent compendia, such as Ph. Eu. and USP fail to pescibe the appopiate method. 8 The detemination of t M is subject to much contovesy. 8 In addition to the uncetainty suounding the calculation of t M, the influence of the stationay phase must also be taken into account. Many liquid chomatogaphic (LC) methods ae descibed in the Ph. Eu. and USP. These methods often use evesed-phase (RP)-LC C8 columns. Howeve, the desciption of an LC method in the Ph. Eu. o USP neve mentions the band of stationay phase that can o must be used. Some infomation on columns that povide adequate sepaations is available though othe channels, such as Phameuopa. Howeve, because hundeds of diffeent RP-LC C8 column bands ae available, diffeent columns will be selected fo the same analysis. Does RRT emain constant when the same analysis is pefomed on diffeent column bands? One might think that, even if etention times vay on diffeent columns, the RRT does not. Howeve, this is not tue. Identification based on the RRT may lead to difficult o incoect peak assignments, esulting in uncetainty of esults. Imagine the consequences if impuities ae misidentified in a chomatogaphic method that applies diffeent impuity limits. The same poblem occus in methods in which peak aeas must be coected, depending on diffeences in specific absobances. In these instances, incoect peak identification can lead to inaccuate content deteminations and/o incoect decisions egading compliance of the substance examined. This study focuses on uncetainty elated to use of the RRT when sepaations ae pefomed on diffeent stationay phases. Five sepaations, pescibed by the Ph. Eu., wee pefomed on 9 diffeent RP-LC C8 columns. Fo each sepaation, the RRT values fo all components wee calculated and the intecolumn vaiability studied. Expeimental Samples: Buflomedil hydochloide, -(pyolidin--yl)--(- hydoxy-,-dimethoxyphenyl)butan--one (impuity A) and -(pyolidin--yl)--(-hydoxy-,-dimethoxyphenyl)butan- -one (impuity B) wee obtained fom the Ph. Eu. laboatoy (Stasboug, Fance). Chloamphenicol and chloamphenicol disodium dissuccinate wee puchased fom the Ph. Eu. and chloamphenicol sodium succinate fom Pham-Inte (Bussels, Belgium). Nimesulide, N-(,-dinito-- phenoxyphenyl)methanesulphonamide (impuity A), N-(-phenoxyphenyl)methanesulphonamide (impuity B), -phenoxyaniline (impuity C), -nito--phenoxyaniline (impuity D) and N,N-bis(methylsulphonyl)--phenoxyaniline (impuity E) wee obtained fom the Ph. Eu. laboatoy. Acetylsalicylic acid (ASA), salicylsalicylic acid (SSA), -hydoxybenzoic acid (HBA), -hydoxyisophthalic acid (HIPA) and acetylsalicylsalicylic acid (ASSA) wee obtained fom Acos Oganics (Beese, Belgium), and salicylic acid (SA) was puchased fom Meck (Damstadt, Gemany). Acetylsalicylic anhydide (ASAN) was pepaed in the laboatoy accoding to a descibed method. 9 Lincomycin, lincomycin B, 7-epilincomyin and 7-epiclindamycin wee obtained fom Phamacia Upjohn (Kalamazoo, Michigan, USA), and clindamycin fom Alpha Phama (Zwevegem, Belgium). Imagine the consequences if impuities ae misidentified in a chomatogaphic method that applies diffeent impuity limits. Chomatogaphy: Analyses wee pefomed using a Vaian (Walnut Ceek, Califonia, USA) 9 LC pump, 9 autosample and 9 UV-VIS detecto, with ChomPefect. softwae (Justice Laboatoy Softwae, Fife, UK) fo data acquisition. Column tempeatue was maintained by immesion in a wate bath. The injection volume was µl in all analyses. The analyses of buflomedil, chloamphenicol, acetylsalicylic acid, nimesulide and clindamycin wee pefomed in accodance with the coesponding Ph. Eu. monogaph. All solvents and eagents wee of Ph. Eu. quality. The 9 stationay phases used ae listed in Table. Fo each sepaation, columns that did not comply with the pesciption of the Ph. Eu. monogaph o accoding to the esults of the system suitability test (SST) wee eliminated. The mobile-phase composition was kept constant fo all columns, as was the flow-ate of. ml/min. No attempts wee made to impove the sepaations by adapting the composition. Duing peliminay expeiments uacil was used as a make fo the hold-up time. A mg/ml solution of uacil in mobile phase was injected thee times befoe each sample analysis. Chomatogaphic conditions fo the diffeent sepaations ae summaized below: Sepaation of buflomedil: The mobile phase consisted of acetonitile 9. g/l potassium dihydogen phosphate adjusted to ph. with phosphoic acid (/ v/v). The column was maintained at C and the detection wavelength was nm. The sample contained buflomedil (9% m/m), impuity A (%) and impuity B (%), and mg of this sample was dissolved in ml of mobile phase. Sepaation of chloamphenicol: The mobile phase consisted of methanol phosphoic acid wate (// v/v/v). The column was maintained at C and detection was pefomed at 7 nm. The sample contained chloamphenicol sodium succinate (9% m/m), chloamphenicol (%) and chloamphenicol disodium disuccinate (%), and. mg of this sample was dissolved in ml of mobile phase. Chloamphenicol sodium succinate is a mixtue of chloamphenicol--sodium succinate and chloamphenicol-- sodium succinate. Sepaation of nimesulide: The mobile phase consisted of acetonitile. g/l ammonium dihydogen phosphate adjusted to ph 7. with ammonia (/ v/v). The column was maintained at C and detection was at nm. The sample contained nimesulide (9% m/m), impuity A (%), impuity B (%), impuity C (%), impuity D (%) and impuity E (%), and mg of this sample was dissolved in ml of acetonitile wate (/ v/v). Sepaation of acetylsalicylic acid: The mobile phase consisted of phosphoic acid acetonitile wate (// v/v/v). The column was maintained at C and the detection wavelength was 7 nm. The sample solution contained. mg of HBA,. mg of HIPA,. mg of SA,. mg of ASA,. mg of ASSA,.7 mg of SSA and. mg of ASAN in ml of acetonitile. The sample was pepaed daily because some of the compounds ae unstable in solution.

3 Table : Stationay phases involved in this poject. Column Name of the column Length Paticle Manufactue/Supplie Type of End- Base Pola Poe numbe (mm)* size (µm) silica capped deactiv. embed. size ACE C8 Advanced Chom. Tech./Achom new ACE C8 Advanced Chom. Tech./Achom new Alltima C8 Alltech new Alltima C8 Alltech new Apex Basic Jones Chomatogaphy/Sopachem old Apex ODS II Jones Chomatogaphy/Sopachem old 7 Aqua Phenomenex/Beste new 8 µbondapak Wates old 9 Bava BDS Alltech new Bava BDS Alltech new Chomolith Meck new - Discovey C8 Supelco new 8 Genesis C8 Jones Chomatogaphy/Sopachem new Hypesil BDS ThemoQuest old Hypesil ODS ThemoQuest old HyPURITY Elite ThemoQuest, SeCoLab new 7 HyPURITY Elite ThemoQuest, SeCoLab new 8 Komasil (MN) Macheey-Nagel/Filte Sevice new 9 Komasil (EKA) Akzo Nobel/SeCoLab new LiChosphe Meck old Luna Phenomenex/Beste new Nucleosil Macheey-Nagel/Filte Sevice old Nucleosil HD Macheey-Nagel/Filte Sevice new Nucleosil Nautilus Macheey-Nagel/Filte Sevice new OmniSphe Vaian new Pecosphe C8 8 PekinElme new 8 7 Platinum C8 Alltech new 8 Platinum C8 Alltech new 9 Platinum EPS C8 Alltech new Platinum EPS C8 Alltech new Podigy Phenomenex/Beste new Puosphe Meck new 8 Puosphe endcapped Meck new 8 Puosphe STAR e Meck new 8 SPHERI- PekinElme new 8 Spheisob ODS Wates old 8 7 Supelcosil LC-8 Supelco old 8 Supelcosil LC-8 DB Supelco new 9 Supelcosil LC-8 DB Supelco new Supesphe Meck new Symmety Wates new TaceExcel ODS A- Teknokoma/SeCoLab NA NA NA NA TaceExcel ODS A- Teknokoma/SeCoLab NA NA NA NA TSKgel ODS-8TS TosoHaas/SeCoLab new 8 TSKgel Supe ODS TosoHaas/SeCoLab new Uptisphee HDOC8 Intechom/Achom new LC GC Euope Novembe

4 Table : (continued). Column Name of the column Length Paticle Manufactue/Supplie Type of End- Base Pola Poe numbe (mm)* size (µm) silica capped deactiv. embed. size 7 Uptisphee HDOC8 Intechom/Achom new 8 Uptisphee ODB Intechom/Achom new 9 Uptisphee ODB Intechom/Achom new Validated C8 PekinElme new Wakosil C8 HG SGE/Achom new Wakosil C8 HG SGE/Achom new Wakosil C8 RS SGE/Achom new YMC-Hydosphee C8 YMC Sep. Tech./ThemoQuest new YMC-Pack Po C8- YMC Sep. Tech./ThemoQuest new YMC-Pack Po C8- YMC Sep. Tech./ThemoQuest new 7 Zobax Eclipse XDB-C8 Agilent Technologies new 8 8 Zobax Extend C8 Agilent Technologies new 8 9 Zobax SB-C8 Agilent Technologies new 8 * The intenal diamete is always. mm. Sepaation of clindamycin: The mobile phase consisted of acetonitile.8 g/l potassium dihydogen phosphate adjusted to ph 7. with a g/l solution of potassium hydoxide (/ v/v). The column was maintained at C and the detection wavelength was nm. The clindamycin hydochloide sample contained clindamycin (88.% m/m), lincomycin (.%), lincomycin B (.9%), 7-epilincomyin (.%) and 7-epiclindamycin (.%), and mg of this sample was dissolved in ml of mobile phase. Each chomatogam was ecoded thee times. Mean RRT values wee calculated using the fomula = (t R t M ) / (t R(st) t M ), whee t R is the etention time of the component of inteest, t R(st) the etention time of the standad and t M the hold-up time. The standad o efeence peak coesponded to the substance to be examined. Results The fist sepaation shown is that of buflomedil. The ode of elution is: impuity B, buflomedil and impuity A (Figue ). The monogaph pescibes the use of a. mm column packed with endcapped µm octadecylsilyl silica gel. Howeve, the Ph. Eu. allows adaptation of the column length by 7%, the intenal diamete by % and the paticle size (eduction only) by %. Columns without endcapping (see Table ), column (a monolithic column) and column ( µm paticle size) did not meet these Ph. Eu. equiements and wee emoved. Columns that did not comply with the SST, which asks fo a esolution of at least. between buflomedil and impuity B, wee emoved as well. The RRT values on suitable columns ae shown in Figue. The peak coesponding to buflomedil is taken as the efeence peak ( ). Although the peak identification in this sepaation is simple, it can be noticed that vaies fom.7 to. fo impuity B and fom. to.98 fo impuity A. Column shows a coelution of buflomedil and impuity A. A typical chomatogam of the sepaation of chloamphenicol sodium succinate is shown in Figue. The elution ode of this sepaation is: chloamphenicol, chloamphenicol--sodium succinate, chloamphenicol-- sodium succinate and chloamphenicol-,-disodium disuccinate. Results fo the analysis ae shown in Figue. The Ph. Eu. monogaph pescibes the use of a. mm Figue : Typical chomatogam of buflomedil on column 7; RRT values fo the sepaation of buflomedil and its impuities fo the columns that comply with Ph. Eu. equiements Peaks: ( ) impuity B, ( ) buflomedil, ( ) impuity A. Column numbe

5 column packed with µm octadecylsilyl silica gel. To comply with the SST, the peaks coesponding to chloamphenicol and chloamphenicol disodium disuccinate must be clealy sepaated fom the two peaks coesponding to chloamphenicol--sodium succinate and chloamphenicol-- sodium succinate. All columns pesented in Figue comply with these equiements and only thee columns wee emoved. The peak coesponding to chloamphenicol--sodium succinate was taken as efeence peak ( ). Compaed with the pevious sepaation, a somewhat lage vaiation in can be obseved. Fo the chloamphenicol peak, vaies fom.7 to.78 and fo the peak coesponding to chloamphenicol--sodium succinate fom.7 to.9. These anges ovelap, indicating a seious poblem fo peak identification when based on the RRT alone. Indeed, peaks with an RRT of aound.7 may coespond to chloamphenicol o chloamphenicol--sodium succinate, depending on the stationay phase used. As the Ph. Eu. limits the chloamphenicol content to.%, but does not limit the content of chloamphenicol--sodium succinate, a clea identification is impotant. Theefoe, the Ph. Eu. monogaph pescibes the use of a chloamphenicol chemical efeence substance (CRS) solution to identify this peak. The RRT of the peak coesponding to chloamphenicol-,-disodium disuccinate vaies fom.9 to.. A thid analysis is that of ASA. On most RP-LC C8 columns the ode of elution is: HBA, HIPA, ASA, SA, ASSA, SSA and ASAN. A typical chomatogam is shown in Figue. The monogaph pescibes the use of a. mm column packed with µm octadecylsilyl silica gel. To comply with the SST, the esolution between the ASA and SA peaks must be at least. Figue shows the RRT values fo all columns (8 out of a total of 9) that comply with these equiements. The peak coesponding to ASA was taken as the efeence peak ( ). Fo HBA, vaies fom. to.8 and fo HIPA fom. to.. On column, HBA coelutes with HIPA. Column shows a coelution of HIPA and ASA. If columns and ae eliminated, the anges become HBA :.. and HIPA :... The RRT fo SA vaies between.9 and.98, fo ASSA between.7 and.7, fo SSA between. and., and fo ASAN between.9 and.7. An ovelap of the adjacent anges above is obseved fo all these impuities. Although this is the esult of some columns behaving vey diffeently, it does show how caeful one should be with the identification of peaks based on the RRT alone. When. would be pescibed as an indicative RRT fo SSA, an analyst using column could identify ASSA as SSA, while an analyst using column could identify ASAN as SSA. It should be noticed that the stationay Figue : Typical chomatogam of chloamphenicol sodium succinate on column ; RRT values fo the sepaation of chloamphenicol--sodium succinate and its impuities fo the columns that comply with Ph. Eu. equiements Column numbe Figue : Typical chomatogam of ASA on column ; RRT values fo the sepaation of ASA and its impuities fo the columns that comply with Ph. Eu. equiements Column numbe 7 Peaks: ( ) chloamphenicol, ( ) chloamphenicol--sodium succinate, ( ) chloamphenicol--sodium succinate, ( ) chloamphenicol-,-disodium disuccinate. Peaks: ( ) impuity HBA, ( ) impuity HIPA, ( ) impuity ASA, ( ) impuity SA, ( ) impuity ASSA, ( ) impuity SSA, 7( ) impuity ASAN. LC GC Euope Novembe

6 phases that give somewhat diffeent RRT values fo this sepaation ae not the same as those that give diffeent RRT values fo the sepaation of chloamphenicol. A typical chomatogam of the sepaation of nimesulide is shown in Figue. On most LC-RP C8 columns, nimesulide and its impuities ae eluted as follows: impuity A, nimesulide, impuity B, impuity C, impuity D and impuity E. Results fo the sepaation of nimesulide ae pesented in Figue. Fo this sepaation, the Ph. Eu. pescibes the use of a. mm column packed with octadecyl silica gel. The SST asks fo a minimum esolution of. between the two peaks coesponding to impuity C and D. The columns pesented in Figue comply with these equiements. Fo impuity A, vaies fom to., fo impuity B fom.7 to.8, fo impuity C fom.9 to.7, fo impuity D fom. to. and fo impuity E fom. to.7. An ovelap of anges is obseved fo impuities B and C, impuities C and D, and impuities D and E. This leads to difficult peak identification based on the RRT alone. An RRT of. may coespond to impuity C o D, while an RRT of. may coespond to impuity D o E, depending on the stationay phase used. Moeove, the elution ode on column 9 has changed. Although this column shows sufficient esolution between impuity C and D and thus passes the esolution test, the elution ode of impuities D and E is Figue : Typical chomatogam of nimesulide on column ; RRT values fo the sepaation of nimesulide and its impuities fo the columns that comply with Ph. Eu. equiements Column numbe Peaks: ( ) impuity A, ( ) nimesulide, ( ) impuity B, ( ) impuity C, ( ) impuity D, ( ) impuity E. evesed. Theefoe, an identification of the peaks, based on the RRT alone, will esult in incoect peak identification. Again, this shows the impotance of the use of chemical efeence substances. The Ph. Eu. monogaph pescibes the use of nimesulide impuity C CRS and nimesulide impuity D CRS. Indeed, these ae citical peaks egading peak identification. The sepaation method used fo clindamycin was based on the Ph. Eu. monogaph, which is a monogaph that uses RRT values in the SST. The USP uses the same chomatogaphic method. A typical chomatogam is shown in Figue. The monogaph pescibes the use of a. mm column packed with µm octadecylsilyl silica gel. Columns Figue : Typical chomatogam of clindamycin on column ; RRT values fo the sepaation of clindamycin and its impuities; (c) a/b values fo clindamycin and its impuities fo the columns that comply with Ph. Eu. equiements. (c) Column numbe Column numbe Peaks: ( ) lincomycin B, ( ) lincomycin, ( ) 7-epi-lincomycin, ( ) clindamycin B, ( ) 7-epi-clindamycin. 7

7 and wee emoved as they did not comply with these equiements. Both Ph. Eu. and USP epot elative etentions with efeence to clindamycin, fo impuity A (lincomycin) about., fo impuity B (clindamycin B) about. and fo impuity C (7-epi-clindamycin) about.8. One would expect RRT values fo diffeent columns to be distibuted andomly aound these values. Howeve, when RRT values wee calculated, not one column showed an RRT aound. and vey few aound. and.8 (Figue ). Moeove, when the esults in Figue ae examined, it can be obseved that the RRT values fo lincomycin B, lincomycin and 7-epilincomycin show lage vaiations when analysed on diffeent stationay phases. The RRT values fo lincomycin B vay fom to., fo lincomycin fom. to. and fo 7-epi-lincomycin fom. to.8. In this sepaation a peak with. may coespond to thee diffeent impuities, depending on the stationay phase used. The RRT values fo clindamycin B vay fom. to. and fo 7-epi-clindamycin fom.7 to.8. The diffeence between the pescibed and the expeimentally obseved RRT values found is most likely the esult of the fomula used. Indeed, it is supposed that the RRT values pescibed in the Ph. Eu. and USP texts wee obtained using Equation, which does not take into account the holdup time. Theefoe RRT values wee also calculated using this equation. Results ae shown in Figue (c). It can be obseved that a/b values ae much bette distibuted aound the values indicated in the monogaph. When the esults pesented in Figue (c) ae examined, the a/b values fo lincomycin B vay fom. to., fo lincomycin fom. to.9, fo 7-epi-lincomycin fom. to., fo clindamycin B fom.8 to.78, and fo 7-epi-clindamycin fom.78 to.878. This coesponds bette to the pescibed RRT. Still, it emains clea that it is not possible to identify the peaks using the RRT only. Such discepancies between the fomula pescibed in the geneal pat of the Ph. Eu. and the values in the monogaphs can be assumed to exist in many monogaphs. This is because the RRT fomula was adapted when the th edition of the Ph. Eu. was published, while the RRT values in the monogaphs wee not. It is still possible that many chomatogaphes use Equation to calculate the RRT, and epot esults that may then be used to daft official monogaphs. This is pobably why the poblem is also encounteed in the USP, although it has been using Equation fo some time. Conclusion These expeiments show that epoducibility of RRT values is uncetain on diffeent stationay phases. Theefoe, peak identification based on RRT alone may lead to misjudgements. It was also shown that inadequate use of fomulas can lead to vey diffeent esults. To make the official fomulas fully applicable, it is necessay that instuctions ae given fo the detemination of hold-up times. This study was pefomed on 9 diffeent column bands, but in a maket offeing moe than column bands, the poblems ae obviously even geate. Given the fact that diffeent limits may be pescibed fo the diffeent impuities in a substance examined, and that impuity suface aeas often have to be coected, the unambiguous identification of peaks is of majo impotance. The use of appopiate chemical efeence substances o the use of a efeence sample, spiked with impuities, togethe with a typical chomatogam is in many instances the only way fo peak identification with sufficient cetainty. Acknowledgements The authos thank the manufactues and the supplies fo the gifts of columns. Refeences. R.E. Majos and P.W. Ca, LC GC N. Ame., 9(), ().. United States Phamacopeia, United States Phamacopeial Convention Inc., Rockville, Mayland, USA ().. Euopean Phamacopoeia d edition, Euopean Depatment fo the Quality of Medicines, Stasboug, Fance ().. Euopean Phamacopoeia edition, Euopean Depatment fo the Quality of Medicines, Stasboug, Fance ().. M.J.M. Wells and R. Clak, Anal. Chem.,, (98).. A.M. Kstulovic, H. Collin and G. Guiochon, Anal. Chem.,, 8 (98). 7. H. Engelhadt, H. Mülle and B. Deye, Chomatogaphia, 9, (98). 8. C.A. Rimme, C.R. Simmons and J.G. Dosey, J. Chomatog. A, 9, 9 (). 9. H. Bundgaad and C. Bundgaad, J. Pham. Phamacol.,, 9 98 (97).. Euopean Phamacopoeia, Addendum., Euopean Depatment fo the Quality of Medicines, Stasboug, Fance ().. D. Visky et al., Phameuopa,, (). Piete Dehouck is a PhD student at K.U. Leuven, Laboatoy of Phamaceutical Chemisty and Dug Analysis, Leuven, Belgium, woking in the field of uncetainty detemination in phamaceutical analysis and column classification. Doa Visky is a post-doc eseache fom the Semmelweis Univesity, Depatment of Phamaceutical Chemisty, Budapest, Hungay, woking in the field of column classification, LC MS and CE MS. Zsuzsanna Kovács is a PhD student at Semmelweis Univesity, Depatment of Phamaceutical Chemisty, Budapest, Hungay, using sepaation techniques (HPCE, HPLC), electoanalytical and spectoscopic (NMR, UV) methods to chaacteize and analyse dugs and biomolecules. Béla Noszál is Pofesso of and Head of Phamaceutical Chemisty at Semmelweis Univesity, Budapest, woking in the fields of phamaceutical and biomolecula analysis and micospeciation. Ewin Adams is a post-doctoal fellow of the Fund fo Scientific Reseach Flandes (Belgium), actually woking at K.U. Leuven, Laboatoy of Phamaceutical Chemisty and Dug Analysis, Leuven, Belgium. He is mainly woking in the field of LC MS and column classification. Jos Hoogmatens is Pofesso of and Head of the Laboatoy of Phamaceutical Chemisty and Dug Analysis, K.U. Leuven, 8 LC GC Euope Novembe