Capillary Electrochromatography of Complex Plasma Matrix on a C18/SCX Column Using UV-Vis and Mass Spectrometric Detection 9 1" V. Splkmans / S. J. Lane 2 / N. W. Smith 1
1Zeneca / SmithKline Beecham Centre for Analytical Sciences, Imperial College of Science, Technology and Medicine, South Kensington, London, SW7 2AY, UK 2Glaxo Wellcome R&D, Gunnels Wood Road, Stevenage, Herts, SG1 2NY, UK
capillary High Performance Liquid Chromatography (cap LC), combining the advantages of both separation methods. The flow through the column is electrically driven, resulting in a fiat flow profile9 Due to this plug shaped flow profile, high efficiencies with good sensitivity and selectivity are typical [1-16]. Using mass spectrometric (MS) detection in combination with CEC offers further enhancement in selectivity [ 17-26].
Key Words Capillary electrochromatography Mass spectrometry Mixed mode columns Plasma analysis
Summary Separations of compounds in plasma were performed on a Hypersil Duet C18/SCX capillary electrochromatography (CEC) column, utilizing an automated injection system combined with a short in-house designed and fabricated micro-electrospray CEC mass spectrometer interface. Protein precipitation was used prior to the CEC separations. More than two hundred separations of the corticosteroids Dexamethasone and Betamethasone 17-valerate, and Fluticasone Propionate in complex plasma matrix were performed on a single column under isocratic conditions. The method demonstrated good reproducibility, selectivity, sensitivity and high efficiencies. Linear calibration with good correlation was typical. Estimated detection limits in the low micromolar and nanomolar range for all compounds were obtained using UV-Vis absorbance (UV) and Electrospray Mass Spectrometric (ES/MS) detection respectively. Efficiencies for all compounds were typically 87,500 plates on a 25 cm column (350,000 plates m -1) and increased with the number of plasma samples injected, up to 250,000 plates per column (1,000,000 plates m<). These very high observed plate counts may be artificially enhanced by the inadequate scan possibilities of the MS over very narrow chromatographic peaks.
Introduction Capillary Electrochromatography (CEC) is a new high efficiency separation technique, which can be considered as a hybrid between Capillary Electrophoresis (CE) and 18 0009-5893/00/01 18-07 $03.00/0
In order to compete with other commercially available methods, such as HPLC, CEC has to demonstrate its compatibility in a wide range of application fields. The applicability of CEC in biofluid analysis is a key area but is presently considered problematic9 To date successful CEC analysis ofbiofluids has involved time-consuming and/or complicated sample pre-treatment protocols. Only a few articles have demonstrated CEC separations of plasma samples [27-29]9 For the separation of corticosteroids in plasma, Taylor et al. used dialysis in combination with gradient CEC for trace enrichment. Paterson et al. and Stead et al. reported the essential use of solid phase extraction (SPE) prior to the CEC separation of plasma samples. Due to the small dimensions used in CEC, most problems using complex matrices are thought to arise due to (irreversible) absorption and subsequent blockage of the column [27]9At present, it is rather difficult to give an explanation for these blockages, which may be due to the amount and/or size of the proteins and/ or the amount of ions present in the application samples. In this article, a Hypersil Duet Cls/SCX mixed mode column is used for the separation of the corticosteroids Dexamethasone and Betamethasone 17-valerate and Fluticasone Propionate in the complex plasma matrix. The difference between this column and the mixed mode columns utilized in previous articles [23-26, 30] is in the aromatic linker used for the bonding of the apolar chains to the silica particles and the ultra pure silica used as the backbone for the stationary phase. The compatibility of CEC with samples containing plasma matrix is illustrated under isocratic conditions, utilising only protein precipitation prior to the separations. Again, the robust automated injection system for capilary electrochromatography combined with a short in-house designed and fabricated micro-electrospray CEC/MS interface, is used in combination with MS detection [23, 24].
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Table I. CalibrationofDexamethasone,FluticasonePropionateand Betamethasone17-valeratein plasma, usingUVand MS detection(8 data points for each calibration).Column:25 cm • 100 #m i. d. 3/~mHypersilDuetC18/SCX;Separationvoltage:16kV (UV), 17 kV (MS);Injection 15 kV for 10 s; Mobilephase: 80 % CH3CN/ 25 mM NHaOAcpH 4.0; Detection:230 nm (UV); Appliedpressure: 10 bar both vials (UV). Compound Slopeand intercept Dexamethasone FluticasonePropionate Betametbasone
UV-Visdetectioncalibration r2 Concentration Range(/~M)
y= 1.08x- 0.26 y=2.72x+0.57 y = 0.47x+ 1.41
0.992 0.999 0.994
3.80-244.80 0.63- 40.00 3.60 - 286.70
Experimental Material Aqueous solutions were prepared, using distilled and deionized (18Mohm) ultra pure water from an Elga Maxima waterpurifier. Acetic acid was purchased from BDH Laboratory Supplies (Poole, UK) and ammonium acetate from Fisons (Loughborough, UK). The corticosteroids (Dexamethasone and Betamethasone 17-valerate) were obtained from Sigma Chemical Co. (St. Louis, USA) and Fluticasone Propionate (FP) is a Glaxo Wellcome compound, used for the treatment of seasonal rhinitis. All chemicals were of analytical grade. Acetonitrile and methanol were purchased from Rathburn Chemicals Ltd. (Walkerburn, Scotland). 40 cm of which 25 cm packed • 375 #m o. d. • 100 #m i.d. 3 #m Hypersil Duet C18/SCX (Strong Cation Exchange) columns were purchased from Hypersil (Runcorn, UK). The columns were refritted and cut to size as required, using a capillary former purchased from Innovatech Ltd. (Stevenage, UK).
C E C / U V a n d MS A Hewlett Packard 3DCE instrument (Hewlett Packard, Waldbronn, Germany) was used for performing CEC with UV-Vis absorbance detection. Both buffer vials were pressurized up to 10 bar during the separations. Injections were performed by applying 15 kV for 10 seconds and separations were performed at 16 or 20 kV, depending on the current generated in the column. The mobile phase consisted of 80% CH3CN / 2 5 m M NH4OAc (pH 4.0) in all experiments. The measurements were performed at 230 nm. After the CEC/UV measurements, the CEC capillary was cleaved at the terminating frit and installed into the CEC/MS system. CEC/MS was performed on a Finnigan MAT TSQ 7000 triple quadrupole mass spectrometer (San Josr, California, USA), using an in-house designed/fabricated micro-electrospray (ES) CEC/MS interface [23]. The capillary was heated up to 150 ~ and the spray voltage was 2.8 kV. A sheath flow of 2.0 #L min -t 70% methanol/water + 0.1% acetic acid was supplied by a Hewlett Packard model 1050 HPLC pump
ES/MSdetectioncalibration r2 Concentration Range(/~M)
y= 1.89E+06x-2.11E + 06 y= 1.79E+06x-3.32E+05 y = 1.24E+06x- 3.43E + 06
18-07 $ 03.00/0
0.999 0.999 0.987
0.60-76.50 0.10- 12.50 0.57 - 71.70
(Waldbronn, Germany), using a 1/100 Acurate splitter (LC Packings, Amsterdam, The Netherlands). Injections and separations were performed using an inhouse newly designed automated injection system [24]. The injection and separation voltages were applied by a high voltage unit (Alpha III model 3807, Brandenburg Ltd., Thornton Heath, UK). The mobile phase consisted of 80 % CH3CN / 25 mM NH4OAc (pH 4.0) and the separation voltage was 20 kV in all experiments. Injections were performed at 15 kV for 10 seconds.
Sample Pre-treatment The proteins in 100 #L dog plasma are precipitated by addition of 300#L acetonitrile. The sample is vortex mixed afterwards and centrifuged at 13,000 rpm for 5 minutes. The decanted supernatant was blown to dryness under nitrogen at 37 ~ and the samples were reconstituted in 200 #L water/acetonitrile (50/50 (v/v)), containing the compounds Fluticasone Propionate, Betamethasone 17-valerate and Dexamethasone in different concentrations (from nM to #M). Injections were performed directly from the reconstituted sample. According to literature, 99 % or more of the proteins are precipitated, using this procedure [31 ].
Results and Discussion Calibration in Plasma Matrix Calibration lines in the #M range were constructed for Dexamethasone, Fluticasone Propionate and Betamethasone 17-valerate in plasma matrix using UV detection (Table I). Greater sensitivity using ES/MS detection allowed a wider concentration range from nM to #M (Table I). Equal but non optimised conditions were used with ES/ MS as with UV detection to demonstrate the feasibility of CEC/MS. All calibration lines are correlated to peak areas instead of peak heights, as diffusion has a relatively large influence on short sample plugs, compared with long sample plugs, during the CEC separation [32]. The results were linear over the range measured for both detection methods, as shown in Table I. The lowest levels analysed with UV detection, i. e. 3.8, 0.6 and 3.6 #M for Dexamethasone, Fluticasone Propionate and
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TableI1. Efficiencies,retentiontimesand observedcurrentsfor the meas~ementsofthe analyticalstandardsamplebeforeand afterthe plasma measurements,using UV detection.The conditionsused were as described in Figure 1. D = Dexamethasone,FP = FluticasonePropionate and B = Betamethasone17-valerate. Measurement (Separationvoltage,kV)
Efficiency(platesm-1) D FP
B
Beforeplasma(20 kV) Plasma(16 kV)
253,000 272,000
361,000 366,000
364,000 384,300
Afterplasma(20 kV)
283,000
398,000
414,000
Betamethasone 17-valerate respectively, demonstrate that the system is comparably sensitive to general CEC/ UV systems. The detection limits of the same compounds in analytical samples were in the identical range, also indicating that the plasma matrix has no detrimental effect on the sensitivity of the system. The calibration lines illustrate that plasma samples can be analysed in a very reproducible manner by CEC on the Hypersil Duet Cls/SCX columns, using UV as well as ES/MS detection. Only protein precipitation is required for compatibility with small-dimension CEC. When increasing the concentrations of the compounds using ES/MS detection up to approximately 100 #M and higher, a plateau was reached for the MS signal for the Dexamethasone peak areas. This upper limit and consequential deviation from linearity with higher concentrations may result from incomplete ionisation of the compounds during the electrospray process, causing the divergence in peak area measured. Optimisation of the electrospray conditions through addition of sheath gas and/or reducing the sheath flow rate may enhance the ionisation o f the compounds, facilitating a wider dynamic range. The same difficulty was encountered, using analytical samples, containing the compounds in concentrations of 100#M and higher in combination with ES/MS detection. Plasma and analytical standard samples of 100/~M and higher were successfully measured, using UV detection, without reaching a plateau in the signal for the areas of the compounds measured. These results suggest that it is unlikely that the plasma matrix is responsible for the deviation obtained. Betamethasone 17-valerate however could be analysed in the MS at all concentrations introduced, even at concentrations higher than 100 ~M. Considering the structural similarities between Betamethasone 17-valerate and Dexamethasone, optimised electrospray conditions would be expected to be similar. The divergence in peak area at higher concentrations of Dexamethasone injected cannot be totally explained by incomplete ionisation during the electrospray process. The compounds do not contain a protonation site and Atmospheric Pressure Chemical Ionisation (APCI) may improve ionisation efficiency. Nevertheless, samples in trace analysis will generally contain concentrations much lower than 100 #M of the compounds of interest. 20
Retentiontime(rain)-+RSD D FP
B
Current(#A) +RSD
5.31 5.68 -+0.18 (n= 8) 5.34
6.97 7.56 -+0.00 (n = 8) 6.97
24.8 21.7 -+1.0 (n = 8) 24.8
6.67 7.23 -+0.28 (n = 8) 6.70
The sensitivity of ES/MS was found to be higher than for UV for the corticosteroids, although these compounds represent a structural class that would not be expected to provide the best electrospray sensitivity. Detection limits (estimated around 500 nM), based on the lowest levels analysed (which were based on signal to noise ratio of ca. 5), were expected to be poorer for the corticosteroids than achieved previously for salbutamol and salmeterol (low nM) [26]. The corticosteroids have no protonation site, while salbutamol and salmeterol are totally ionized. The difference in detection limit may be partly due to this. A further contributing factor is that for the same electrokinetic injection conditions different amounts will be injected dependent on the electrophoretic mobility of the sample constituents. The electrophoretic mobility of salbutamol and salmeterol will be higher than of the uncharged corticosteroids. This difference in mobility would normally be compensated using off-line UV detection by the peak traversing the cell faster than the uncharged species. This is not the case with ES/MS detection. Due to the same injection method used in both the salbutamol/ salmeterol experiments and the experiments performed here, the amount injected in the experiments with the basic compounds might be higher, due to the superimposing effect of the electrokinetic mobility. Therefore, lower detection limits might have been obtained for the salbutamol and salmeterol measurements, compared to the detection limits obtained in these experiments.
Reproducibility of Plasma Analysis, Using UV Detection Figure 1 A and C demonstrate the separation reproducibility of an analytical standard sample containing the three compounds before and after one hundred plasma separations, using UV detection. Figure 1 B shows an example of the electropherogram obtained from the separation of the compounds in a plasma sample, following the protein precipitation with acetonitrile. All measurements were performed without reconditioning of the column in between measurements. Compound 4 is a plasma contaminant, clearly separated from the compounds of interest. Table II summarizes retention times and efficiencies obtained for the different compounds for the experi-
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Plasmaimnple
Sample before plasma
A
1 5.31
mAU 80
B
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25
Sampleafter plasma
1
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A and C showUV-electropherogramsof the same analytical sample, containing 306.0/~MDexamethasone (1), 50.0/~M Fluticasone Propionate (2) and 286.7/~M Betamethasone 17-valerate (3), before and after the measurement of plasma samples respectively. B shows an example of the electropherograms obtained when analyzing a plasma sample (containing 122.4/~M Dexamethasone, 20.0pM Fluticasone Propionate and 114.8/~M Betamethasone 17-valerate). Peak 4 is a plasma contaminant. A and C were performed using a separation voltage of 20 kV, while B was performed at 16kV. Further conditions were the same as described in Table I.
ments illustrated in Figure 1. This includes the observed current through the column during measurements, used as a diagnostic for potential column blockages. Highly reproducible retention times and selectivities are achieved for all three compounds during all measurements, even after the separation of more than approximately one hundred plasma samples on the same column. The RSD of the retention times for the different compounds in plasma are below 0.3 % (n = 8). Shorter retention times are obtained in electropherograms A and C, compared to B, due to the higher separation voltage applied. A higher current was observed through the column during the plasma separations, resulting in the obligatory use of a lower separation voltage to avoid air bubble formation and column breakdown, even when pressurizing the in- and outlet vials. Using MS detection, columns can be run at much higher currents, without having to pressurize the in- and outlet vials (100 #A and higher through a 100#m i.d. • 30cm column). Joule heating is never observed using these conditions with MS detection. The current was also very stable during all measurements. Even after the plasma measurements the current remained constant, suggesting that no plasma matrixinduced column blockage occurred. As shown in Table II, high efficiencies are obtained for all three compounds in all experiments performed. The efficiencies for the compounds during the plasma seOriginal
parations are higher than the efficiencies obtained for the analytical sample before these plasma measurements. The efficiency is enhanced after the first plasma sample is introduced on the column and is subsequently increased every time a plasma sample is injected and separated. The efficiencies of the compounds in the analytical sample after the plasma measurements are higher, compared to the analytical sample before the plasma separations and are even higher than the efficiencies obtained during the plasma measurements. An explanation for this behavior of the efficiencies can not be given at present, but a similar effect was observed using other, new Hypersil Duet C]8/SCX columns. A possibility might be that the frits are conditioned, due to compound(s) present in plasma. It is unlikely that changes have taken place inside the column itself, since very stable retention times and selectivities were observed during all measurements. The heated frit is a key and contentious part of the column and its activity has been the subject of much discussion [33]. The results obtained here might indicate that the frits have activity that can be eliminated. The activity could be serious to low compound levels and for basic compounds. The key would be a derivatisation procedure to completely deactivate thermal frits. Another explanation for the high efficiencies might be the use of the aromatic linkers and/ or the ultra pure silica in the Hypersil columns, Nevertheless, this will only explain the high efficiencies generally obtained for this type OfCl s/SCX column and will
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A
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before ~um8 5.81
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IIIIIFI~IrlIIIFllItIIIrHIIl[l~"klllllIIIgllIIIIpIIII,lllllrl[n IIIIIll]MI]Itlr] %0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 0.0
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r r r v ~ . . r ~ . i - w ~ r ~ . r ' m r r r - r l - r wl..~.lVp ~ 2,0 4.0 6.0 8.0 10.0 ~lme (rain)
Figure 2 A and C show MS-electropherograms of MH+-ions for the compounds in an analytical sample before and after the measurement of plasma samples (containing 306.0 #M Dexamethasone, 50.0 #M Fluticasone Propionate and 286.7 #M Betamethasone 17-valerate). B shows an example of the electropherograms obtained when measuring a plasma sample (3.8 #M Dexamethasone, 0.6 #M Fluticasone Propionate and 3.6 pM Betamethasone 17-valerate). The conditions used were the same as described in Table I. The compound with a retention time of 3 min. is an impurity present in all samples. The ion counts are shown in the right upper comers. m/z = 399.3: Dexamethasone, 501.2: Fluticasone Propionate and 477.3: Betamethasone 17-valerate.
Table III. Efficiencies, retention times and currents for the plasma measurements and the measurements of the analytical sample before and after the plasma measurements, using MS detection. The conditions used were as described in Figure 2. D = Dexamethasone, FP = Fluticasone Propionate and B = Betamethasone 17-valerate. Measurement (Separation voltage, kV)
Efficiency(plates m 1) D FP
Retention time (min) D FP B
Current (#A)
B
Before plasma (20 kV) Plasma (20 kV) After plasma (20 kV)
250,000 279,300 817,000
330,000 423,700 1,300,000
5.81 5.80 5.74
24 24 24
310,000 349,100 1,090,000
not explain the increase in efficiency when introducing plasma samples on the column. The effect was found to be reversible and after blank injections, efficiencies would return to their original plate numbers.
A c c o r d i n g to these results, highly selective, efficient and reproducible plasma separations are obtainable on the Hypersil Duet C18/SCX CEC columns, using only protein precipitation.
22
7.27 7.17 7.08
7.91 7.70 7.74
Reproducibility of Plasma Analysis, Using MS Detection The same samples used in UV-Vis detection were repeated using ES/MS detection, after the column was cleaved after the terminating frit. The efficiencies, retention times and currents for these measurements are shown in Table III and examples o f the separations are illustrated in Figure 2. The separations were performed at an effective field strength o f 17 kV (20 k V applied by
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Table IV. Efficiencies obtained for the plasma measurements and
the measurements of the analytical sample before and after the plasma measurements, using UV detection. All separations are performed at 16 kV. Further conditions used were equal to the conditions described in Table I. D = Dexamethasone, FP = Fluticasone Propionate and B = Betamethasone 17-valerate. Measurement
Beforeplasma Plasma First afterplasma Secondafterplasma
Efficiency(platesm -1) D FP
B
285,500 356,800 300,500 277,900
264,300 341,400 308,900 274,900
255,000 373,500 302,500 258,500
high voltage power supply unit minus 3 kV electrospray voltage). The compound with m / z 501.2, eluting at approximately 3 minutes, is an impurity present in all samples. The peak is present in plasma and in analytical samples. Highly reproducible retention times and selectivities are obtained for all compounds with ES/MS detection, even after the analysis of the plasma samples. Approximately one hundred plasma samples were once again separated on the same colunm, in between the analytical samples measured. Due to buffer depletion, the retention times gradually increased with the number of measurements performed [24, 26]. However, after refreshment of the buffer, the retention times were equivalent to the retention times obtained before all measurements. The current remained very stable during all measurements as well, indicating that no blockages arise from the plasma matrix being analysed. The efficiencies obtained during the plasma measurements are enhanced when compared with the efficiencies obtained in the analytical samples before the plasma separations. Also the efficiencies of the analytical sample after the plasma measurements are higher than the efficiencies obtained during the plasma analysis. However this effect is more outstanding in the ES/MS detection than in the UV detection measurements. The efficiencies of the three compounds in the analytical sample, measured after the plasma samples, are even around 200,000 to 325,000 plates on the 25 cm column (800,000 to 1,300,000 plates m 1). These very high observed plate counts may be artificially enhanced by the inadequate scan speeds of the MS over very narrow chromatographic peaks. Only 4 to 5 points for peaks, having peak widths at half peak height of around 2 seconds, were obtained using a scan speed of 2 scans per second. The efficiencies obtained for the compounds in the analytical sample, analysed before the plasma separations with ES/MS detection are lower compared to the efficiencies obtained in the analytical samples after the plasma separations using UV detection. This indicates that the enhancement in efficiency is a reversible effect. To confirm these observations, the same measurements were performed on a new Hypersil Duet C18/SCX colOriginal
umn, to demonstrate that the effect is present in every column. The results are displayed in Table IV. The efficiencies generally obtained are lower than in the previous measurements. This was thought to result from a poorly packed column. Nevertheless, the results confirmed the reversible nature of the effect. The efficiencies of the compounds used, increase when a plasma sample is introduced and separated on the column. The first sample, immediately analysed after the plasma sample, still showed increased efficiencies, compared to the same analytical sample injected before the plasma separation. The efficiencies are down to normal levels again when the second analytical sample is injected and separated after the plasma analysis. This clearly demonstrates that the plasma matrix used, induces an enhancement in efficiency, although in a reversible manner. Further research on this phenomenon is currently under investigation.
Conclusions All measurements in this article (except for the measurements in Table IV) were performed on a single Hypersil Duet C18/SCX column under isocratic conditions demonstrating the potential of CEC for the high resolution analysis of complex plasma matrix and the robustness and reproducibility of the CEC/MS system. No difficulties in using the column were observed, even though ultra pure silica is used. No loss in efficiency and selectivity is observed either during the separation of plasma samples, when compared to analytical samples and the retention times of the compounds remain very stable during and after the measurement of plasma samples. More than two hundred samples were separated in a reproducible manner on a single column. The effficiencies appear enhanced after every plasma sample introduced on the column. This may result from in situ conditioning of the column or more likely the frits by plasma components. Further experiments are needed to enable control of this in an irreversible manner. The current in the column remains very constant as well, during and after the plasma measurements, indicating that no problems were encountered due to blockage of the column.
Acknowledgements The authors are grateful to Rob Plumb from Glaxo Wellcome Ware for the kind donation of the dog plasma. We would like to acknowledge Zeneca and SmithKline Beecham for funding the analytical centre at Imperial College. This work was supported by a PhD scholarship from Glaxo Wellcome Stevenage.
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[23] S. J Lane, A. Pipe, Rapid Commun. Mass Spectrom., 12, 667 (1998). [24] S. J Lane, M. G. Tucker, Rapid Commun. Mass Spectrom., 12, 947 (1998). [25] R.N. Warriner, A.S. Craze, D.E. Games, S.J. Lane, Rapid Commun. Mass Spectrom., 12, 1143 (1998). [26] V. Spikmans, S. J Lane, U.R. Tjaden, J van der Greef Rapid Commtm. Mass Spectrom., 13, 141 (1999). [27] M. R. Taylor, P. Teale, S. A. Westwood, D. Perrett, Anal. Chem., 69, 2554 (1997). [28] C.J. Paterson, R.J. Bought[lower, D. Higton, E. Palmer, Chromatographia, 46, 599 (1997). [29] D.A. Stead, R. G. Reid, R.B. Taylor, J. Chromatography A, 798, 259 (1998). [30] N. W. Smith, 34. B. Evans, J. Chromatography A, 832, 41 (1999). [31] J. Blan chard, J Chrom (Biomed Apps), 226, 455 (1981) [32] M. Mazereeuw, U R. Tjaden, J. van der Greef J. Chromatography A, 677, 151 (1994). [33] N. W.Smith, "The effect of the retaining frits on chromatographic performance in Capillary Electrochromatography", 23ra International Symposium on High Performance Liquid Phase Separations and Related Techniques, Granada, Spain, 1999.
Chromatographia Vol. 51, No. 1/2, January 2000
Received: Jun 1, 1999 Revised mansucripts received: Jul 16 and Aug 16, 1999 Accepted: Sep 16, 1999
Original
