Anal. Chem. 1994,66, 1090-1096
Quantification in Capillary Zone Electrophoresis for Samples Differing in Composition from the Electrophoresis Buffer Joachlm Leube and Odette Roeckel' Pharma Division, Preclinical Research, Hoffmann-La Roche Ltd., CH-4002 Basel, Switzerland
Quantitative precision and accuracy in capillary zone electrophoresis (CZE) with electrokinetic (EK)injection is drastically improved by using matrix-corrected peak area (COPA) combined with calibration by regression with one internal standard. COPA compensates for the differentially changing electrophoretic mobilities of analytes and internal standards in sample matrices differing in composition from the electrophoresis and/or calibration buffer. Intraassay precision and accuracy are improved by factors up to 12 and 6.5, respectively. For interassay conditions, the inaccuracy data are lowered from beyond 15%to significantly below 5% relative deviation from the nominal concentration value. The simulation of biological samples was achieved by using 12 sample buffers differing in pH, conductivity, and viscosity, whereas the separation buffer was kept constant. Due to the efforts of a continuously increasing number of researchers, for example, Mikkers, Everaerts, and Verheggen1g2 and Jorgenson and L u k a c ~capillary ,~ electrophoresis (CE) has become accepted as a highly efficient separation method for qualitative purposes. Special techniques such as micellar electrokinetic chromatography (MECC)k9 and capillary gel electrophoresis1&12have been developed to extend the field of application. So far, however, CE has not been established as a quantification method, probably due to the uncontested predominance of chromatographic methods in the field of quantitative analysis. Furthermore, there is still a lack of confidence in CE with respect to its quantitative capabilities. Some of the workers dealing with quantification in capillary zoneelectrophoresis (CZE) and MECC point out problems (1) Mikkers, F. E. P.;Everaerts, F. M.; Verheggen, Th. P.E. M. J . Chromarogr. 1979, 169, 1-10. (2) Mikkers, F. E. P.; Everaerts, F. M.; Verheggen, Th. P.E. M. J . Chromarogr. 1979,169, 11-20. (3) Jorgenson, J. W.; Lukacs, K. D. AMI. Chem. 1981,53, 1298-1302. (4) Fujiwara, S.; Honda, S. Anal. Chem. 1987, 59,2773-2776. (5) Weinbcrger, R.; Albin, M.J . Liq. Chromatogr. 1991, 14, 953-972. (6) Otsuka, K.; Terabc, S.; Ando, T. J. Chromatogr. 1987, 396, 350. (7) Fujiwara, S.;Iwase, S.; Honda, S. J . Chromatogr. 1988, 447, 133. (8) Burton, D. E.; Sepaniak, M. J.; Maskarinec, M.P.J . Chromarogr. Sci. 1986, 24, 347. ( 9 ) Nishi, M.; Tsumagari, N.; Kakimoto, T.; Tertabe, S. J. Chromatogr. 1989, 477, 259. (10) Cohen, A.; Karger, B. J . Chromatogr. 1987, 397, 409-417. (1 1) Cohcn, A.; Nagarian, D.; Smith, J.; Karger, B. J . Chromatogr. 1988, 458,
323-333. (12) Cohcn, A.; Paulus, A.; Karger, B. Chromarographia 1987, 24, 15-24. (13) Huang, X.;Luckey, J. A.; Gordon, M. J.; Zare, R. N. Anul. Chem. 1989.61,
766-770.
(14) Tsuda, T.; Mizuno, T.; Akiyama, J. Anul. Chem. 1987, 59, 799-800. (15) Tsuda, T.; Nomura, K.; Nakagawa, G . J . Chromafogr. 1983,264,385-392. (16) Tsuda, T.; Nakagawa, G.;Sato, M.; Yagi, K. J . Appl. Biochem. 1983, 5, 330-336. (17) Fujiwara, S.;Honda, S.Anal. Chem. 1986, 58, 1811-1814. (18) Roach, M. C.; Gozel, P.;Zare, R. N. J . Chromarogr. 1988, 426, 129-140. (19) Lookabaugh, M.; Biswas, M.; Krull, I. S.J . Chromatogr. 1991,549,357-366. (20) Liu, J.; Cobb, K. A.; Novotny, M. J. Chromatogr. 1989, 468, 55.
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Analytical Chemistry, Voi. 66, No. 7, April 1, 1994
concerning precision and accuracy, and it turns out that electrokinetic (EK) sample injection is more critical than hydrodynamic (HD) injection. On the other hand, EK injection exhibits the advantage of easy handling, and the velocity discrimination of the sample during loading may improve selectivity. EK injection, which appears at first glance to be under the same degree of control as H D injection, is actually influenced by many experimental factors, including injection voltage, injection time, and surface chemistry of the capillary wall. The total amount of a charged solute loaded onto the capillary is contributed from both its electrophoretic migration velocity in the sample buffer and the electroosmotic plug flow of the electrolyte system used for ~ e p a r a t i o n . ~ For * , ~ this ~ reason, precision and accuracy in EK injection are greatly affected by sample parameters. Even small variations of relevant parameters in the sample solution may alter the effective mobility peffof the solute during the injection period, thus leading to a bias in peak area.30 Recently, a new quantification method for CE with EK injection31 has been introduced, which compensates for conductivity differences in the electrophoresis buffer and the sample buffer, whereas other sample parameter biases are not corrected for. On the other hand, internal standardization methods utilizing one4,6J3J7,32 or two internal standards35put large constraints on the selection of the internal standard. In addition to the criteria valid also for HPLC analysis, the optimum internal standard in CZE/EK should be almost identical electrophoretically to the analyte. This implies, first, that the migration times are distinct but very similar and, second, that the dependency of the electrophoretic mobilities on the sample parameters should be very similar for analyte and internal standard. This is a prerequisite that cannot easily be met with complex pharmaceutical and biological samples of differing and/or unknown composition where matrix effects on the mobilities may vary from one samule to another. (21) Cobb, K. A.; Novotny, M. Anal. Chem. 1989, 61, 2226.
(22) Huang, X.; Coleman, W.; Zare, R. J. Chromatogr. 1989, 480, 95. (23) Bocek, P.;Dcml, M.;Gebauer, P.; Dolnik, V. Anulyrical Isorachophoresis; VCH: Weinheim, Germany, 1988. (24) Issaq, HH.; Atamna, I.; Muschik, G.;Janini, G. Chromarographia 1991,32, 155. (25) Altria, K. D.; Simpson, C. F. Chromatographia 1987, 24, 527. (26) Altria, K. D.; Simpon, C. F. Anal. Proc. 1988, 25, 85. (27) Bruin, G.J. M.; Chang, J.; Kuhlman, R.; Zegers, K.;Kraak, J.; Poppc, H. J . Chromatogr. 1989, 471, 429. (28) Nashabch, W.; Rassi, Z. E. J. Chromatogr. 1990. 514, 57. (29) Adamson, A. W.Physical Chemistry of Surfaces, 4th ed.;Wiley Interscience: New York, 1981. (30) Huang, X.; Gordon, M.J.; Zare, R. N. Anul. Chem. 1988, 60, 375-377. (31) Lec, T.T.; Yeung, E. S.Anal. Chem. 1992,64, 1226-1231. (32) Honda, S.;Iwase, S.;Fujiwara, S. J . Chromatogr. 1987, 404, 313-320. (33) Wallingford, R. A.; Ewing, A. G.Adu. Chromarogr. 1989, 29, 1. (34) Hueckcl, E. Physik. Z . 1924, 25, 204. (35) Dose, E. V.; Guiwhon, G.A. Anal. Chem. 1991, 63, 1154-1158. 0003-2700/94/036&1 OB0$04.50/0 0 1994 American Chemical Society
In this paper, we present a new method of calibration by regression using matrix-corrected peak areas (COPA). This COPA method corrects for all sample parameters and thus significantly improves both precision and accuracy for all sample types tested. Twelve different sample solutions were used to model the situation in bioanalysis, where the biological matrix may vary from sample to sample. The effect of sample pH, electrical conductivity, and viscosity on the peak areas of analyte and internal standard was systematicallyinvestigated on a manual CE device. In addition, our investigations on an automated CE apparatus were subjected to a comparative study of the EK injection using the new COPA method and of the HD injection using uncorrected peak areas for quantification.
THEORY The effective electrophoretic mobility pcff of an ion is determined not only by the solution parameters P H , ionic ~~ strength I (conductivity ~ ) , and 2 viscosity ~ ~ ~ 7 , but also by the substance parameters effective charge qefr29and hydrodynamic radius r,z9which in return depend on the composition of the solution. For pH-sensitive weak electrolytes,p,ff(pH) is proportional to their degree of dissociation a:23 pcfl(pH) = ap- for weak acids
(1)
pefl(pH) = ap+ for weak bases
(2)
where pcff(pH)is the electrophoretic mobility as a function of the pH, and p - and p+ are the mobilities of the anion and cation, respectively. For not too extreme solutions, the mobility of an ion pcn(Z) depends inversely on the ionic strength P4according to eq 3:
(3) where pcn(Z) is the electrophoretic mobility as a function of the ionic strength of the solution, p o is the mobility at infinite dilution (I = 0), and k is a constant for the respective ion. According to DebeyeHueckel, for small ions, the effective charge qcfr decreases with increasing ionic strength of the solution. The cloud of counterions becomes denser, thus shielding the original charge on the central ion more effectively. In addition, during migration an extra viscous force is exerted on the central ion, which is stronger for a dense ion cloud. For spherical particles, a generally valid expression for p C ~which , comprises all parameters, is given by the following equations: 29,34
Pcff
4crr = 6?rqr
where qcff is the effective chargez9 of the ion, r is its hydrodynamic radius, 7 is the viscosity, c is the dielectric constant of the solution, and fis the f-potential of the solute.34 For ions of similar charge, r increases with the charge density of thecentral ion, i.e., smaller ions are more strongly hydrated, thus leading to lower pcff as compared to larger ions. For similar reasons, r increases with the charge number of the
solute, f was found to be inverselyproportional to the amount of ions present at the surface of the solute. In return, the number of ions is partly determined by the pH and the ionic strength of the solution. We aimed at a bias factor that compensates for all sample parameters. The most reliable measure for the influence of sample matrix parameters on pen of a solute during injection and, thus, on the total loaded amount of the respective analyte is the apparent peak area. For this reason, we define the matrix factor Furl as a normalized bias factor that describes the change of peak area of an analyte in a sample matrix relative to that in a defined reference matrix of similar analyte concentration (eq 6):
where PA,, is the peak area of analyte i in the sample matrix x, and P&li is the respective peak area of analyte i in the sample solution used for calibration. For the calculation of valid Furl, blank sample matrices or simulated sample matrices have to be spiked with the respective analyte at a defined concentration. On the condition that FMxi is constant over the entire calibration range, matrix-corrected peak areas can be determined from the peak area raw data obtained for the respective sample matrix (eq 7): PArxi COPA,, = FMxi
(7)
where COPA,i is the matrix corrected peak area of analyte i in the sample matrix x, PArxi is the actual peak area raw data of analyte i in the sample matrix x, and F Mis ~the~ respective matrix factor calculated according to eq 6. While the term PA,i refers to the peak area of an analyte in a sample of known concentration, which is used to determine its Furi, PA,i represents the peak area of an analyte obtained from an unknown sample. The use of COPA instead of PA for analytes and internal standard improves quantitative precision and accuracy data.
EXPERI MENTAL SECT I ON The manual Bio-Rad (Richmond, CA) HPE 100 capillary electrophoresissystem and a 50-pm4.d. fused silica capillary (Bio-Rad), total length 50 cm (47.2cm effective length), were used for systematic investigations. Additionally, the automated CE apparatus AB1 270 A HT (Applied Biosystems, Forster City, CA) with a 50-pm4.d. and 70 cm total length (50 cm effective length) capillary was employed for a comparative study of EK and HD injection. The separations were performed at ambient temperature and at a constant temperature of 30 OC, respectively. The on-line UV detector was operated at 230 nm; for both devices, recording and integration of the peaks were performed by a Spectra Physics SP 4200 integrator (Spectra Physics, San Jose, CA). The electrophoresis buffer consisted of a 40 mM borate buffer, pH 9.5,and an electrical conductivity K of 1756 p S / cm. Twelve sample electrolytes were used to model varying biological matrices, made up of the same buffer system mentioned above, but differing in pH, electrical conductivity K (25 "C), and viscosity 9 (25 "C): (1) pH 9.0 ( K = 1098 pS/cm); (2)pH 9.3 ( K = 1420pS/cm); (3) pH 9.5( K = 1756 AnaMical Chemisby, Vol. 88, No. 7, April 1. 1994
'1091
Table 1. Schwnatlc Presentation ot Data Evaluattkn.
intraassay day 1 day 2 day 3 interassay
c1 c1 c1
RSDl
c2 c2
c3 c3
CZ RSDz
C3 RSD3
C4 c4 c4
RSDd
CS CS CS RSDS
CS CS c6
RSDs
c8
c7 c7
c7
RSD,
CS CS RSDS
c9 c9
CS RSDs
ClO ClO ClO RSDlo
cl2 el2
c 1 1
e11 c11
RSDll
C12 RSDlz
RSDA RSDB RSDc
C1-12 = determined concentrations of analytes for the individual sample buffers 1-12. Table 2. Intraaamy. Ouantltatlve Preclrlon* and Inaccuracyc (IACC) wlth Manual EK Injection Urlng Peak Area (PA) and Matrlx-Corrected Peak Area (COPA) (Manual Devlce BbRad HPE 100)
1
11.4 9.2 8.4 3.1 13.4 9.3 9.0 5.8 8.1 6.5 6.2 2.1
PA RSD analyte 2 3 8.2 5.9 5.3 2.9 9.8 5.4 6.5 4.6 6.0 3.7 3.8 2.6
5.3 3.8 3.7 1.8 6.4 3.8 4.1 3.4 4.0 2.7 2.7 1.9
COPA
IACC analyte 1
2
3
1
RSD analyte 2
9.6 6.5 13.4 4.0 11.3 6.6 14.6 6.8 6.8 4.5 9.8 2.1
6.8 4.0 10.1 3.0 8.0 3.6 11.1 5.1 5.0 2.6 7.7 2.4
4.3 2.4 6.4 1.8 5.3 2.6 1.4 3.3 3.3 1.7 4.8 1.6
1.0 0.9 0.4 1.3 2.7 1.0 1.3 2.3 2.9 1.9 1.5 1.8
0.7 0.8 0.2 0.8 2.1 0.5 2.5 1.3 1.9 1.3 1.3 0.7
3 0.5 0.4 0.2 0.7 1.4 0.4 0.6 1.1 1.2 0.1 0.8 0.6
1
IACC analyte 2
3
1.5 1.1 1.8 1.6 2.4 1.1 3.2 2.1 2.4 1.1 2.6 2.5
1.1 1.7 2.3 2.2 2.0 1.4 3.0 1.1 1.9 1.9 1.1 2.7
0.9 0.7 1.0 1.0 1.4 0.9 2.1 0.8 1.1 0.8 0.9 1.4
0 Intraaeaay conditions as given in Data Evaluation; 12 sample buffers differing in pH, electrical conductivif and viscosity. In %RSD. Determined as the mean % deviation from the nominal analyte concentration of 1.25 mM on the basis of 12 ifferent samples. Precision and inaccuracy data are determinedon the basis of the five sam le buffers with different pH values. e RSD and IACC determined on the basis of sample buffer of pH 9.5 and four NaCl buffers. f RSD and IA8C determined on the basis of sample buffer of pH 9.5and three HPMC buffers.
pS/cm); (4) pH 9.7 ( K = 2030pS/cm); (5) pH 10.0 ( K = 2270 FS/cm); (6) pH 9.5, 17 mM sodium chloride ( K = 3710 pS/ cm); (7) pH 9.5,34 mM sodium chloride ( K = 5780 pS/cm); (8) pH 9.5, 51 mM sodium chloride ( K = 6840 pS/cm); (9) pH 9.5,68 mM sodium chloride ( K = 8700 pS/cm); (10) pH 9.5,O.l%HPMC (hydroxypropylmethylcellulose) ( K = 1689 pS/cm, 9 = 1.72 cP); (11) pH 9.5, 0.2% HPMC ( K = 1685 pS/cm, 9 = 2.22 cP); (12) pH 9.5, 0.4%HPMC ( K = 1755 pS/cm, I] = 4.12 cP). N-Acetyltyrosine (analyte l ) , hippuric acid (analyte 2), N-acetylbenzoic acid (analyte 3), benzoic acid (internal standard), and acetophenone (neutral marker) were of analytical grade. The internal standard and neutral marker concentrations were constant at 2.5 and 10 mM, respectively. Calibration was carried out with analyte concentrations of 0.25, 0.5, 1.0, 2.0, and 2.5 mM. Samples contained 1.25 mM of each analyte. The separation voltage was set to 8 kV (22 kV, automated device), EK injection was performed at 6 kV for 5 s (1 5 kV for 5 s, automated device), and HD injection was performed with the AB1 270 A H T at 5 mmHg vacuum for 2 s. At the beginning of each analysis day, a l-h conditioning run with the electrophoresis buffer was performed. Cleaning and equilibration of the capillary between the individualruns was achieved with two intermediate rinsing steps using 100mM NaOH and electrophoresis buffer, respectively. Conductivity and pH measurements were performed with a WTW LF 521 conductivity detector and a WTW LDM/S conductivity cell (WTW Lauda, Germany), and a microprocessor pH-meter 762 (Knick, Berlin, Germany), respectively. The viscosity measurementsof the HPMC-containing sample buffer solutions were done with an ‘Ostwald’ viscosity apparatus. 1092 Ana&?icalChemistry, Vol. 66, No. 7, April 1, 1994
Data Evaluation. Calibration and sample application were carried out once on each of three consecutivedays. Intraassay variation is defined as the relative standard deviation (RSD) calculated from the analyte concentrations determined for the 12 sample buffers on one day (dependency of RSD on sample matrix). Interassay refers to the RSD values obtained for the three concentrations at constant sample buffer on the three consecutive days of analysis ( 12 RSDs). Accuracy data were obtained on the basis of mean percentage deviations from the nominal sample concentration. The two different tests are schematically shown in Table 1. This data evaluation scheme is identical to the data evaluation performed routinely for bioanalysis in our laboratories. RESULTS AND DISCUSSION Since the injection period is very short relative to the separation period and the length of the sample zone is very small considering the total length of the capillary, injection contributions to migration time variance are very small as compared to separation contributions. For this reason, intraassay precision of migration times (see Table 6) is better for automated than for manual EK injection. Essentially, this is due to the efficient heat dissipationduring theseparation phase in the automated device. On the other hand, as a result of the immense interplay of the various experimental parameters associated with EK injection, intraassay precision with traditional calibration by regression using PA (see Tables 2 and 4) is poor for both manual and automated injection. Therefore, we were not successful in our first attempt using migration time-corrected peak area (PA/&,) instead of PA for quantification (data not shown). In contrast, the use of matrix-corrected peak areas (COPA; see eq 7) eliminates the
Table 3. Interassay' Preclslonb and InaccuracyC(IACC) wlth Manual EK Injectlon Using Peak Area (PA) and Matrix-Corrected Peak Area (COPA) (Manual Device Blo-Rad HPE 100)
PA
COPA
RSD analyte sample buffer
IACC analyte
1
2
3
1
9.0 9.3 9.5 9.7 10.0
3.0 2.4 1.0 0.6 1.0
2.5 1.8 0.4 0.4 0.7
1.4 0.9 0.3 0.3 0.5
15.3 12.4 1.1 3.4 6.6
17 mM 34 mM 51 mM 68 mM
2.3 3.4 4.1 4.4
1.8 2.6 3.0 2.7
1.4 1.7 2.1 1.7
12.8 15.5 16.8 16.8
0.1% 0.2 % 0.4 %
4.0 3.2 1.9
2.8 2.1 1.0
1.9 1.3 0.8
7.4 6.0 2.6
RSD analyte
2 ,
IACC analyte
3
1
2
3
1
2
3
7.5 5.6 0.7 0.7 1.4
2.6 2.4 1.0 0.6 1.0
2.0 1.8 0.4 0.4 0.7
1.5 0.9 0.3 0.3 0.8
1.9 1.5 1.1 0.9 1.0
1.9 1.8 1.7 1.7 1.6
1.0 0.8 0.7 0.8 0.8
5.8 7.3 8.6 8.7 HPMC 6.0 4.4 4.6 3.1 1.7 0.7
2.3 1.2 4.1 4.3
1.7 2.6 3.0 2.7
1.3 1.7 2.0 1.7
2.0 2.8 3.3 3.6
1.2 2.4 2.8 2.5
1.2 1.5 1.8 1.5
1.2 3.2 1.9
2.8 2.1 1.0
1.9 1.3 0.8
3.3 2.6 1.3
2.6 2.0 1.6
1.7 1.2 0.6
10.1 8.2 1.7 1.0 2.7
PH.
NaCl 9.3 11.7 12.3 13.1
Interassay conditions as given in Data Evaluation; 12 sample buffers. b In % RSD. c Determined as the mean % deviation from the nominal
analyte concentration of 1.25 mM on the basis of three days of analysis.
Table 4. Intramsay. Quantltatlve Precisionb and InaccuracyC (IACC) with EK InJgctlen Uslng Peak Area (PA) and Matrix-Corrected Peek Area (COPA) and wlth HD Injection Using PA (Automated Devlce ABI)
EK injection PA COPA RSD
IACC
day1 day2 day2
11.8 11.8 10.3
9.1 9.2 8.1
day 1 day 2 day3
10.0 9.4 8.5
7.2 6.0 6.3
day 1 day2 day3
5.0 4.8 3.1
4.5 4.5 5.5
RSD
HD injection PA
IACC
RSD
IACC
1.3 1.6 1.8
2.2 1.9 3.9
2.0 1.8 3.1
1.5 2.6 0.9
2.8 3.1 1.8
2.3 2.5 1.8
1.4 3.0 2.4
2.1 2.9 3.2
1.6 2.8 2.7
1.4
1.2
matrix factor
t I
Analyte 1
1.4 1.4 2.1 Analyte 2 2.0 3.8 0.6 Analyte 3 2.0 3.6 3.2
a Intraassay conditions as given in Data Evaluation; four sample buffers: 40 mM borate buffer (a) pH 9.5, (b) pH 10.0, (c) pH 9.5,51 mM NaC1, (d) pH 9.5,0.2% HPMC. In %RSD. Determined as the mean % deviation from the nominal analyte concentration of 1.25 mM.
930
9,3
9,5
997
10,o
pH of sample buffer
main source of error in EK injection, because the F M x i are a direct measure for changes in sample buffer composition and other influences on peak area. Matrix-Corrected Peak Area: The COPA Method. The F M x i of the different sample buffers were calculated for the three analytes, the internal standard, and the neutral marker according to eq 6. For this purpose, sample solutions were prepared with 2 mM of each analyte, 2.5 mM of internal standard, and 10mM of neutral marker in the different sample buffers. The calibration solution (pH 9.5) of similar concentration was taken as the referencematrix. Since 11 sample buffers were used, which differed from the calibration buffer, 11 matrix factors were determined for the individual species. In order to make sure that there was no dependency on the analyte concentration or on the total solute concentration in the sample solution, the F M x i were also calculated on the basis of 0.25,1.25, and 2.5 mM analyte concentration. The relative
1 +analvte 1
f- analvte2
8analvte 3
I-standard 0N-markerb
Figure 1. Matrix factors Fw of analytes 1,2, and 3, internal standard
(I, standard), and neutral marker (N marker) as a function of sample buffer pH.
standard deviations for concentrationeffects were in the range of 0.3-1% for analytes, internal standard, and neutral marker. Plots of the matrix factors as a function of pH, conductivity (molar content of NaCl), and viscosity (76 content of HPMC) are given in Figures 1-3, and a typical electropherogram is depicted in Figure 4. As can be seen from Figure 1, the F M x i of analytes and internal standard cover the range of about 0.6-1.2 within 1 unit of sample solution pH. Since the sample solution of pH 9.5 was taken as the reference, the correspondingF M x i is 1 for all components. In the applied electric field of positive polarity, Analytical Chemistry, Vol. 66, No. 7, April 1, 1994
1093
1.7
matrix factor
I
1.6
1.5
1.4
1.3
2
1.2
1.1
3
1
0.9
0.8
17
0
34
51
68
mM NaCl of sample buffer
I +analvte 1 +analvte2
8analyte 3
I-standard 8 N-marker
b
Flgure 2. Matrix factors FMxl of analytes 1,2, and 3, internal standard
(I standard), and neutral marker (N marker) as a function of sample buffer conductivity (molar content NaCI). 1.4
i
matrix factor
1.3
I
1.2
1.1
I
1
5
10
Time [ n i n ] Flgure 4. Electropherogram of N = neutral marker (10 mM), 1, 2, 3 = analytes 1, 2, and 3 (1 mM), and I = internal standard (2.5 mM) In the separation buffer of pH 9.5.
11
0.9 0.8
0.7
0.6
0.5
0,o
0,1
02
093
0,4
% HPMC of sample buffer
1 .+analvte 1 +analvte 2 6anaivte 3
I-standard 0 N-marker b
Figure 3. Matrix factors FMxl of analytes 1,2, and 3, internal standard
(I standard), and neutral marker (N marker) as a function of sample buffer viscosity (% content HPMC).
the anionic analytes and the internal standard are migrating in the opposite direction of the electroosmoticflow and, thus, out of the capillary. Therefore, the total amount of the individual species decreases with the increase of their peff. 1094 AnalytlcaIChemistry, Vol. 66, No. 7, April 1, 1994
The significant divergence of the FMxi slopes indicates very different pH sensitivitiesfor analytesand the internal standard, which are most pronounced in the buffers with pH values below 9.5. For intraassay conditions, the PA ratios and, thus, the calculated concentrations significantly differ from the nominal value, thus resulting in high RSDs and inaccuracy data (see Tables 2 and 4). In general, due to the very narrow sample zone, the influence of the sample buffer composition on the electroosmotic flow velocity of the entire electrolyte system during injection is regarded to be negligibly small. In accordancewith this, theFMxiobtained for the neutral marker and thus the injected sample volumes are almost constant over the examined pH range (Figure 1). For the sample buffer of pH 10,which has the highest conductivityof the pH buffers, electroosmoticflow and injected volume are slightly reduced. This observation is corroborated by the data obtained for the high conductivity sample buffers (Figure 2), where injection volumes decreaseby 15-20% with increasing content of sodium chloride. The addition of sodium chloride in the range of 17-68 m M enhances the conductivity by a factor of 2.1-5,
respectively. Obviously, the high ionic strength of the NaClcontaining buffers intermediately leads to a local reduction of the r-potential on the capillary wall and, thereby, decreases the electroosmotic mobility pw of the electrolyte system (see eq 3 which is also valid for p w ) . Similar effects are observed for the pen of analytes and internal standard, which are also inversely related to the ionic strength of the solution. Therefore, in the case of positive polarity, the migration rates into the capillary decrease for cations and increase for anions with increasing sample solution conductivity. Accordingly, and despite the lower applied injection volumes, the FhlXiof the anionic species drastically increases in the NaCl buffers (Figure 2). It is interesting to note that the effect of ionic strength is most pronounced on the way from moderate to high conductivity (0.0-17 mM NaCl). Further addition of NaCl results in a less than proportional rise of the F M x i . In contrast to conductivity, there is no effect of viscosity on the electrical field strength. However, the electroosmotic plug flow and thereby the injected sample volumes are reduced (see Figure 3 and also eqs 4 and 5) in the order of 20-25% (FMxi of neutral marker are from 0.8 to 0.75)in the HPMC buffers. Whereas the penis inversely proportional to viscosity in its entirety, the change in pw is smaller since it concerns only the small contribution from the injection plug. Consequently, the total amount of analytes and internal standard loaded onto the capillary is less in the HPMC buffers as compared to the calibration buffer. But again, as we observed in the case of conductivity, there is a nonlinear relationship between the parameter changed and the respective F M ~ ~ . Intraassay precision and inaccuracy data with manual EK injection are listed in Table 2 for PA and COPA. As could be expected from the slope of the F M (Figure ~ ~ 1-3) by using uncorrected peak areas, precision and accuracy deteriorate with increasing difference in the migration time of analyte and internal standard. For analyte 3, intraassay RSDs are in the range of 4.0-6.5%, and the respective differencesbetween the determined concentrations and the nominal value range from 3.3% to 5.3%. On the other extreme, RSDs of analyte 1 arefrom8,1%to 13.4%,andinaccuracyreachesupto 11.3% deviation from the nominal value. Regarding the relatively ~ and small differences in the corresponding F M of~ analytes internal standard (see Figure 3), lowest contributions to the overall intraassay precision and inaccuracy are fromviscosity. In accordance to the data depicted in Figure 2, inaccuracy is worst for conductivity changes, whereas precision data are in the same order of magnitude as obtained for pH. Relative to the nominal value, the calculated analyte concentrations are systematically lower at increased conductivity and systematically higher at increased viscosity. For this reason, the overall RSDs are higher than the contributions from the individual parameters. The efficiency of our COPA method is indicated by significant improvements of the overall intraassay precision by factors of 3--12. Additionally, inaccuracy data are lowered by factors in the range of 3-6.5. In consideration of what we discussed above, the factors are even higher for the individual contributions from pH and K . Intraassay precision data are now from 0.5 to 1.2% RSD for analyte 3 and from 1.0 to 2.9% RSD in the worst case for analyte 1. On the condition that degradation effects can be neglected, under
Table 5. Interassaya Preckio# and Inaccuracyo(IACC) wlth EK In).ctlon M n g Peak Area (PA) and MatrlxCorrocted Perk Area (COPA) and wlth HD InJection Uahg PA (Automated Devlce ABI)
samplebuffer
EK injection PA COPA RSD IACC RSD IACC
HD injection PA RSD IACC
Analyte 1
pH 9.5 pH 10.0 51 mM NaCl 0.2% HPMC
0.9 0.9 1.4 2.6
pH 9.5 pH 10.0 51 mM NaCl 0.2% HPMC
1.9 1.6 0.5 1.9
pH 9.5 pH 10.0 51 mM NaCl 0.2% HPMC
1.9 1.3 1.2 1.7
0.5 11.0 16.0 7.6
0.9 0.9 1.5 2.5 Analyte 2 1.8 1.9 4.5 1.8 16.5 3.1 0.5 3.2 Analyte 3 2.6 1.9 4.8 1.8 10.3 2.2 1.7 0.7
0.5 1.0 2.6 2.2
1.0 1.3 0.1 1.2
1.3 1.6 1.7 3.9
1.8 1.4 2.9 0.5
0.6 0.8 1.0 1.2
1.8 1.9 2.8 1.9
2.6 2.4 1.4 2.6
1.0 1.1 0.9 1.8
1.9 1.9 2.0 3.2
Interassay conditions as given in Data Evaluation; four sample buffers: 40 mM borate buffer (a) pH 9.6,(b) H 10.0, (c) pH.9.5,61 mM NaCl, (d) pH 9.5,0.2% HPMC. * In % h D . Determlned a~ the mean % deviation from the nominal analyte concentration of 1.25 mM on the basis of three days of analysis. Table 8. Intraassay Preclrlon ( % RSD) of Mlgratlon Timer wlth Manual and Automated EK Injection analyte
neutralmarker day 1 day 2 day 3
3.0 3.2 1.6
day 1 day 2 day 3
1.8 1.5 1.4
1
2
Manual 5.1 5.5 3.5 3.6 2.2 2.3 Automated 2.1 2.5 1.8 2.0 1.8 2.0
3
internalstandard
5.9 3.7 2.4
6.7 2.9 2.7
2.9 2.3 2.2
3.2 2.4 2.4
interassay conditions,the sample buffer composition is constant over all days of analysis. Accordingly, interassay precision (see Table 3) with PA is not improved by the use of COPA but, nevertheless, well acceptable for bioanalytical purposes. However, due to the disproportionate effect of the individual sample buffers on the peak areas of analytes and internal standard, especially with the NaCl buffers, inaccuracies calculated from uncorrected data are extremely high. With COPA interassay, inaccuracy is in the order of 0.6%3.6% deviation from the nominal value, which is equivalent to improvement by factors up to 8.3. These data are very well acceptable for our needs in the quantitative analysis of samples in biological matrices. For comparative reasons, all tests were repeated on the automated device with a reduced set of sample buffer solutions and additionally H D injection was applied. The respective test data (see Tables 4and 5) reveal that EK injectioncombined with our COPA method is at least equivalent to HD injection in terms of precision and accuracy. The limitations of the method are more from the point of practical considerations. The COPA method includes various preliminary investigations. ~ ~ the First of all, the determination of the F M necessitates preparation of simulated sample solutions at defined conAnaiyticalChemistry, Vol. 66, No. 7, April 1, 1994
109s
centrations. Afterwards, twoor three additional runs for each sample matrix have to be performed prior to analysis. Beyond that, concentration effects on the FhlXihave to be excluded within the applied calibration range. However, from what we observed in our studies, concentrations in the lower millimolar range and below are not crucial at all from that point. In pharmaceutical analysis, the availability of blank sample matrices is not a problem. In contrast, the situation in bioanalysis is a little more complicated. The composition of plasma or urine samples from human studies or from animals may differ significantly with the time of sample collection. In this case, the FhlXihave to be determined by standard addition. The respective investigations are at present in progress in our laboratories. Sample solutions are spiked with a large surplus of analyte, relative to the original analyte concentration. As biological samples are in the micro- and nanomolar concentration range, the addition of millimolar amounts of analyte is sufficient, and concentration effects on the FhlXiare unlikely. After all, EK injection is advantageous in many cases, because the selectivity of the separation can be increased with appropriate experimental parameters. Since the matrix factors are directly derived from peak area raw data under experimental conditions similar to those during analysis, any (36) Grushka, E.; McCormick, R. M. J . Chromatogr. 1989, 471, 4 2 1 4 2 8 .
10QS AnatyticaiChemistry, Vol. 86, No. 7, April 1, 1994
ubiquitous or extraneous36 injection related to the individual sample matrix is compensated for. For similar reasons, the selection of the internal standard is also facilitated.
CONCLUSION The described method is very efficient with regard to the compensation of sample buffer effects on errors in quantitative results in CE with EKinjection. Thesignificant improvements are precision and inaccuracy data comparable to those obtained with HD injection, which is a priori superior to EK injection in terms of internal standard controlled quantification. As all an extension to what has been presented so far22~31~33~35 sample buffer parameters, including those related to 'ubiquitous' injection, are taken into account, and thus, even complex sample solutions can be quantified successfully. Additionally, the constraints on an appropriate internal standard are by far lower relative to traditional calibration methods. The application of this new calibration method to samples in biological matrices is currently under investigation. Received for review July 7, 1993. Accepted December 6, 1993.' Abstract published in Advance ACS Abstracts, January 15, 1994.
