165
Clinica Chimica Acto, 78 (1977) 165-172 @ Elsevier/North-Holland Biomedical Press
CCA 8425
A QUANTITATIVE ASSAY OF CORTISOL IN HUMAN PLASMA BY HIGH PERFORMANCE LIQUID CHROMATOGRAPHY USING A SELECTIVE CHEMICALLY BONDED STATIONARY PHASE
J.H.M.
VAN DEN BERG a**, CH.R. MOL a, R.S. DEELDER
aand J.H.H. THIJSSEN
b
a Eindhoven University of Technology, Laboratory of Instrumental Analysis, Eindhoven (The Netherlands) and ’ University Hospital, State University Utrecht, Department of Clinical Endocrinology, Utrecht (The Netherlands) (Received
November
5th, 1976)
Summary The extraction and subsequent liquid chromatographic analysis of human plasma samples for cortisol is described. Extraction and chromatography are optimized, resulting in a recovery for cortisol of 96% and a detection limit of 1 pg cortisol in 100 ml plasma. The application of two chemically modified silicas has been evaluated. The specificity of the method was tested by field desorption - mass spectrometry experiments.
Introduction Adequate information concerning the function of steroid-producing endocrine organs and the interaction of the hypothalamic-hypophyseal system with these organs can be derived from estimations in urine and in plasma. Steroid analyses are applied regularly during diagnosis and control of therapy in cases of hypo-, hyper- and dysfunctions of the adrenal cortex and the gonads. A determination of corticosteroids in human plasma, demands that specificity is combined with high sensitivity. Chromatography fulfils these two conditions. In high performance liquid chromatography these can be realized by using suitable phase systems and efficient columns. At the same time chromatographic parameters and extraction methods should be optimized to determine these low concentrations (about 10 c(g cortisol is present in 100 ml plasma) with reasonable reliability. In addition it is desirable not to overburden the patient by requiring a large amount of blood. The presence of synthetic
* To
whom
requests
for reprints
should
be addressed.
166
steroids like dexamethasone and beclamethasone in plasma should not interfere during the cortisol analysis. The use of liquid-liquid partition mechanisms [ 1,2] has been studied. Dcrivatization of non-chromophoric steroids [3,4] and gradient elution [5] were applied with good results. Meijers et al. [6] used liquid-liquid partition chromatography for the analysis of cortisol but at that time efficient columns were not available, so separation and detection-limit were not optimal. Liquid-liquid systems consisting of dichloromethane, ethanol and water [7,8] appeared to be unstable in time [9,10], so this method is rather unpractical for routine analysis. Liquid-solid chromatography has the disadvantage that the water content of the mobile phase has to be controlled very well, in order not to disturb the separation mechanism by deactivation of the silica. Chemically bonded phases do not have these disadvantages and therefore it seemed useful to develop a liquid chromatographic assay of cortisol in human plasma using a selective chemically bonded stationary phase on silica. Experimental Equipment A Kipp liquid chromatograph (model 771) was used in this study. A Zeiss variable wavelength UV detector (type PM2D LC) was operated at 240 nm. Column and detector cell were thermostatted at ambient temperature by a circulating liquid. In addition a Varian liquid chromatograph (type 8500), equipped with a Chromatronix injection valve (type HPSV 20) and a Varian UV detector (type Variscan) was used. Columns were filled by the balanced-density slurry method. Packing material During the measurement two chemically bonded stationary phases were investigated, in which nitro groups are bonded to the surface of an adsorbent, silica, by chemical reaction. In our laboratory silicagel (LiChrosorb SI 60, Merck) was chemically modified according to a method described by Brust et al. [ll] . Irregular silica, with a mean particle diameter of 5 pm, was chlorinated by thionylchloride. The product was dried and subjected to a reaction with paranitroaniline. During the investigations a similar packing material became commercially available. Nucleosil NO, (Machery and Nagel), a chemically modified spherical silica, was preferred in the later experiments. Chemicals Dichloromethane, propan-2-01 and ethanol, all p.a. grade were purchased from Merck. Sodium hydroxide solutions of 0.25 M were prepared. Standard samples of cortisol and prednisolone were prepared in ethanol at a concentration of 10 ,ug/ml. Dichloromethane for the extraction of the plasma samples was cleaned over an Al,O, column (Woelm, basic, activity grade I).
167
Procedure The extraction of corticosteroids from plasma was carried out with dichloromethane, which was washed over A1203. To 1 ml plasma 7 ml dichloromethane was added and 20 ~1 internal standard (corresponding to 200 ng prednisolone). At the same time 0.1 ml 0.25 M NaOH solution was added to exclude phenolic contaminations from the extract [ 121. This mixture was stirred for 30 set in a vortex mixer. Then the water phase was separated by centrifugation (1500-2000 r.p.m.). 5 ml of the aliquot is transferred and evaporated to dryness under warm nitrogen gas (40°C). The residue is dissolved in 100 ~1 eluent. After 10 min in an ultrasonic bath the sample was homogeneous and could be injected into the liquid chromatograph (60 ~1). Results and discussion Separation and column performance The separation was optimized with a test mixture by varying the mobile phase composition in such a way that the capacity ratio of cortisol was about 5. Cortisol is separated from other substituents in the plasma extract without the loss of to much sensitivity. The selectivity of the chosen phase system, Nucleosil NOz (Machery and Nagel) and modified adsorbent and dichloromethane/propan-2-al/water (97.5 : 2.3 : 0.2, v/v) as eluent is given in Table I. It is practicable to perform analyses of cortisol without interference by dexamethasone. Prednisolone can be used as internal standard. Recently it has been shown that spherical as well as irregular particles can be packed regularly to obtain efficient and reproducible columns with a balanced density-slurry packing procedure. Short elution times and low heights equivalent to a theoretical plate have been achieved. Our columns gave plate heights of 30 pm for cortisol and prednisolone. Sample quantity and detection limit The value of the injection volume is restricted because of the contribution peakbroadening should not exceed certain limits. TABLE
I
CAPACITY Mobile
to
phase:
RATIOS
ON
NUCLEOSIL
NO2
AND
dichloromethaneipropan-2-oIlwater
Compound
P-NITROANILINE (97.5
Capacity
ratio
Modified
silica
MODIFIED
: 2.3 : 0.2,
v/v).
Nucleosil
NO2
11-Deoxycorticosterone
0.37
0.2
Testosterone
0.86
0.3
Corticosterone
1.78
1.04
Cortisone
2.53
2.14
Cortisol
5.73
4.88
Dexamethasone
5.15
8.06
Prednisolone
8.95
8.84
LICHROSORB
SI 60
168
On the other hand the injection volume should be large to ensure a minimal dilution, as has been pointed out by several workers [ 13,141. In this way the injected volume of 60 1.11 was calculated. Our results indicated a minimal detectable amount of 1 ng cortisol. Extraction and recovery Extraction procedures were performed with ethyl acetate, diethyl ether and dichloromethane as solvents. The extraction was judged by means of measuring recovery of cortisol and prednisolone. Cortisol was quantitated by measuring peak heights and correcting against the internal standard. Calibration curves without the extraction procedure were determined with test mixtures; calibration curve including an extraction step with l-ml pool-plasma samples to which increasing cortisol and prednisolone were added, were also determined. The ratio of the slopes of the calibration curves with and without extraction gives us the recovery. Table II gives the recovery of cortisol and prednisolone for the solvents mentioned. From these results it is apparent that the extraction with dichloromethane is to be preferred. Fig. 1 gives the calibration curves for cortisol and prednisolone in standard mixtures and plasma samples for the extraction with dichloromethane. Washing with sodium hydroxide has no significant influence on recovery, but the purity of the sample is increased. Reproducibility The precision of the method has been determined by the mean standard deviation per point of duplicate measurements. For each determination the whole procedure consisting of extraction and chromatographic analysis was carried out. 23 duplicate measurements gave a relative standard deviation of 3.3%. Biological spreading of the cortisol content has been eliminated in this way. Figs. 2 and 3 show the chromatograms obtained with the Nucleosil NO* and nitroaniline-modified silica gel, respectively. Comparison with other methods A critical review comparing several techniques for the measurement of cortisol levels in human plasma will be presented in a separate paper [ 151. In addition three sera were analyzed by HPLC and the eluted cortisol peaks were collected by a micro fraction-collector. The collected samples were analyzed by RIA [ 151 and the results were in very good agreement with the HPLC values.
TABLE
II
RECOVERY Solvent
OF
CORTISOL
AND Recovery (5%)
Ethyl Diethyl
PREDNISOLONE cortisol
FOR
Recovery (%)
acetate
89
82
ether
78
72
96
93
Dichloromethane
SOME
SOLVENTS prednisolone
169
Injected
amount
Cortisol
(ng)
Fig. 1. Calibration curves. o, cortisol test mixtures; a, cortisol extracted mixtures; ., prednisolone added to plasma and extracted from plasma.
from plasma;
0. pradnisolone
test
PREDNISOLONE
CORTISOL
10 TIMF
(min)
5
0
c--
Fig. 2. Chromatogram of plasma dichloromethanelpropan-2-al/water
sample. Column: 200 X 4.6 mm i.d.; Nucleosil NO2 ; dp = 5 pm. Eluent: (97.5 : 2.3 : 0.2. v/v); flow rate 1.15 ml/min. Detection: UV 240 nm.
A
10
20 + TIME (min)
B
C
u-LA_ )
10
20 TIME (min)
-
Fig. 3. Chromatogram of plasma sample: A. without addition of internal standard; B, with addition of internal standard. Column: 220 X 2.1 mm i.d.: modified LiCbrosorb SI 60: dp = 5 pm. Eluent: dichloromethane/propan-2-al/water (97.5 : 2.3 : 0.2, v/v); flow rate 25 ml/h. Detectibn: UV 240 nm. Sample: C. cortisol; P, prednisolone.
171
Fig. 4. Chromatogram
of a plasma sample of a patient
treat&d with dexsmathasone.
Conditions
see Fig. 3.
Positive identifications To show the selectivity of the liquid chromatographic method a chromatogram of a plasma of a patient treated with dexamethasone was made. Fig. 4 shows the result; the cortisol content is decreased drastically by this treatment to 2.5 E.cgcortisol/lOO ml plasma. Fig. 5 demonstrates the results that can be obtained by the off-line coupling of liquid chromatography and field desorption-mass spectrometry (FD-MS). The cortisol was collected as it eluted from the LC column and the wire emitter was submerged in the solution, containing lo-’ g cortisol/ml. The FD-MS of cortisol confirms the identification of the peak in the chromatogram of a plasma extract.
100
a0
no
=o
330
-
Fig. 5. Field desorption-mass a current
of 15 mA; source
spectrum temperature
of cortisol. 75’C.
sso
340
Equipment:
360
370
mb Varian Mat 711;
Emitter
10 f.fm heated
by
172
Acknowledgements We gratefully acknowledge the cooperation of Dr. E.B.M. de Jong, St. Elisabeth Hospital, Tilburg. Thanks are also due to P.J.M.W. Claassen, for technical assistance and Dr. J. van der Greef, University of Amsterdam, for performing FD-MS experiments. References 1
Huber,
J.F.K.,
Meijers,
2
Huber,
J.F.K.,
Alderlieste,
3
Fitzpatrick.
4
Henry,
R.A.,
5
Cavina.
G.,
F.A.
and
Metiers.
7
Hesse,
C..
C.A.M.,
8
Trefz,
F.K.,
Byrd.
9
Panis,
N.A.
(1974)
van den Brust,
O.E..
Berg, L.E.
S. (1973)
J.A.R.J. Hatzel.
and
D.J.
and
12
M&r,
13
Karger.
B.L.,
Martin,
14
Huber,
J.F.K.
(1975)
15
Thijssen.
Anal.
A.
Kochen.
Milley,
W. Sci.
J., Vonk.
I. and Halasz,
and Blanchard,
R.C.
M. and Z. Anal.
Aldercreutz.
J.F.
(1973)
Guiochon. Chem.
N.
J.F.K.
44.
111
Chem.
45,
1337
80.
(1972)
2.
Chem.
Anal.
Klin.
J. Chromatogr.
Sci.
9,
513
89 Chem.
Biochem.
107,
261, 12,
347
193
181
753 and Deelder,
I. (1973) Clin.
R.S.
(1977)
J. Chromatogr.
83,
Chem.
G. (1974) 277.
J. Chromatogr.
Z. Klin.
(1975)
Chem. Anal.
2310
J. Chromatogr.
Huber.
12,
Anal.
H. (1973)
45,
(1971)
(1973)
and
(1972)
Poppe,
Chem.
D. (1974)
J. Chromatogr.
J.H.M..
J.A.R.J.
H. and
and Die&man. Cantafora,
K.
Sebestian,
J.H.H..
Harren,
Hiilsman,
Pietrzik,
10
J.A. and
and Hulsman,
E.T..
Siggia.
Schmit, Moretti,
6
11
C.A.M.
19,
J. Chromatogr.
132.
421
15
710
Anal.
Chem.
46,
1640
341
H. and van den
Berg.
J.H.M.
(1977)
Clin.
Chim.
Acta,
submitted
Clinica Chimica Acto, 78 (1977) 165-172 @ Elsevier/North-Holland Biomedical Press
CCA 8425
A QUANTITATIVE ASSAY OF CORTISOL IN HUMAN PLASMA BY HIGH PERFORMANCE LIQUID CHROMATOGRAPHY USING A SELECTIVE CHEMICALLY BONDED STATIONARY PHASE
J.H.M.
VAN DEN BERG a**, CH.R. MOL a, R.S. DEELDER
aand J.H.H. THIJSSEN
b
a Eindhoven University of Technology, Laboratory of Instrumental Analysis, Eindhoven (The Netherlands) and ’ University Hospital, State University Utrecht, Department of Clinical Endocrinology, Utrecht (The Netherlands) (Received
November
5th, 1976)
Summary The extraction and subsequent liquid chromatographic analysis of human plasma samples for cortisol is described. Extraction and chromatography are optimized, resulting in a recovery for cortisol of 96% and a detection limit of 1 pg cortisol in 100 ml plasma. The application of two chemically modified silicas has been evaluated. The specificity of the method was tested by field desorption - mass spectrometry experiments.
Introduction Adequate information concerning the function of steroid-producing endocrine organs and the interaction of the hypothalamic-hypophyseal system with these organs can be derived from estimations in urine and in plasma. Steroid analyses are applied regularly during diagnosis and control of therapy in cases of hypo-, hyper- and dysfunctions of the adrenal cortex and the gonads. A determination of corticosteroids in human plasma, demands that specificity is combined with high sensitivity. Chromatography fulfils these two conditions. In high performance liquid chromatography these can be realized by using suitable phase systems and efficient columns. At the same time chromatographic parameters and extraction methods should be optimized to determine these low concentrations (about 10 c(g cortisol is present in 100 ml plasma) with reasonable reliability. In addition it is desirable not to overburden the patient by requiring a large amount of blood. The presence of synthetic
* To
whom
requests
for reprints
should
be addressed.
166
steroids like dexamethasone and beclamethasone in plasma should not interfere during the cortisol analysis. The use of liquid-liquid partition mechanisms [ 1,2] has been studied. Dcrivatization of non-chromophoric steroids [3,4] and gradient elution [5] were applied with good results. Meijers et al. [6] used liquid-liquid partition chromatography for the analysis of cortisol but at that time efficient columns were not available, so separation and detection-limit were not optimal. Liquid-liquid systems consisting of dichloromethane, ethanol and water [7,8] appeared to be unstable in time [9,10], so this method is rather unpractical for routine analysis. Liquid-solid chromatography has the disadvantage that the water content of the mobile phase has to be controlled very well, in order not to disturb the separation mechanism by deactivation of the silica. Chemically bonded phases do not have these disadvantages and therefore it seemed useful to develop a liquid chromatographic assay of cortisol in human plasma using a selective chemically bonded stationary phase on silica. Experimental Equipment A Kipp liquid chromatograph (model 771) was used in this study. A Zeiss variable wavelength UV detector (type PM2D LC) was operated at 240 nm. Column and detector cell were thermostatted at ambient temperature by a circulating liquid. In addition a Varian liquid chromatograph (type 8500), equipped with a Chromatronix injection valve (type HPSV 20) and a Varian UV detector (type Variscan) was used. Columns were filled by the balanced-density slurry method. Packing material During the measurement two chemically bonded stationary phases were investigated, in which nitro groups are bonded to the surface of an adsorbent, silica, by chemical reaction. In our laboratory silicagel (LiChrosorb SI 60, Merck) was chemically modified according to a method described by Brust et al. [ll] . Irregular silica, with a mean particle diameter of 5 pm, was chlorinated by thionylchloride. The product was dried and subjected to a reaction with paranitroaniline. During the investigations a similar packing material became commercially available. Nucleosil NO, (Machery and Nagel), a chemically modified spherical silica, was preferred in the later experiments. Chemicals Dichloromethane, propan-2-01 and ethanol, all p.a. grade were purchased from Merck. Sodium hydroxide solutions of 0.25 M were prepared. Standard samples of cortisol and prednisolone were prepared in ethanol at a concentration of 10 ,ug/ml. Dichloromethane for the extraction of the plasma samples was cleaned over an Al,O, column (Woelm, basic, activity grade I).
167
Procedure The extraction of corticosteroids from plasma was carried out with dichloromethane, which was washed over A1203. To 1 ml plasma 7 ml dichloromethane was added and 20 ~1 internal standard (corresponding to 200 ng prednisolone). At the same time 0.1 ml 0.25 M NaOH solution was added to exclude phenolic contaminations from the extract [ 121. This mixture was stirred for 30 set in a vortex mixer. Then the water phase was separated by centrifugation (1500-2000 r.p.m.). 5 ml of the aliquot is transferred and evaporated to dryness under warm nitrogen gas (40°C). The residue is dissolved in 100 ~1 eluent. After 10 min in an ultrasonic bath the sample was homogeneous and could be injected into the liquid chromatograph (60 ~1). Results and discussion Separation and column performance The separation was optimized with a test mixture by varying the mobile phase composition in such a way that the capacity ratio of cortisol was about 5. Cortisol is separated from other substituents in the plasma extract without the loss of to much sensitivity. The selectivity of the chosen phase system, Nucleosil NOz (Machery and Nagel) and modified adsorbent and dichloromethane/propan-2-al/water (97.5 : 2.3 : 0.2, v/v) as eluent is given in Table I. It is practicable to perform analyses of cortisol without interference by dexamethasone. Prednisolone can be used as internal standard. Recently it has been shown that spherical as well as irregular particles can be packed regularly to obtain efficient and reproducible columns with a balanced density-slurry packing procedure. Short elution times and low heights equivalent to a theoretical plate have been achieved. Our columns gave plate heights of 30 pm for cortisol and prednisolone. Sample quantity and detection limit The value of the injection volume is restricted because of the contribution peakbroadening should not exceed certain limits. TABLE
I
CAPACITY Mobile
to
phase:
RATIOS
ON
NUCLEOSIL
NO2
AND
dichloromethaneipropan-2-oIlwater
Compound
P-NITROANILINE (97.5
Capacity
ratio
Modified
silica
MODIFIED
: 2.3 : 0.2,
v/v).
Nucleosil
NO2
11-Deoxycorticosterone
0.37
0.2
Testosterone
0.86
0.3
Corticosterone
1.78
1.04
Cortisone
2.53
2.14
Cortisol
5.73
4.88
Dexamethasone
5.15
8.06
Prednisolone
8.95
8.84
LICHROSORB
SI 60
168
On the other hand the injection volume should be large to ensure a minimal dilution, as has been pointed out by several workers [ 13,141. In this way the injected volume of 60 1.11 was calculated. Our results indicated a minimal detectable amount of 1 ng cortisol. Extraction and recovery Extraction procedures were performed with ethyl acetate, diethyl ether and dichloromethane as solvents. The extraction was judged by means of measuring recovery of cortisol and prednisolone. Cortisol was quantitated by measuring peak heights and correcting against the internal standard. Calibration curves without the extraction procedure were determined with test mixtures; calibration curve including an extraction step with l-ml pool-plasma samples to which increasing cortisol and prednisolone were added, were also determined. The ratio of the slopes of the calibration curves with and without extraction gives us the recovery. Table II gives the recovery of cortisol and prednisolone for the solvents mentioned. From these results it is apparent that the extraction with dichloromethane is to be preferred. Fig. 1 gives the calibration curves for cortisol and prednisolone in standard mixtures and plasma samples for the extraction with dichloromethane. Washing with sodium hydroxide has no significant influence on recovery, but the purity of the sample is increased. Reproducibility The precision of the method has been determined by the mean standard deviation per point of duplicate measurements. For each determination the whole procedure consisting of extraction and chromatographic analysis was carried out. 23 duplicate measurements gave a relative standard deviation of 3.3%. Biological spreading of the cortisol content has been eliminated in this way. Figs. 2 and 3 show the chromatograms obtained with the Nucleosil NO* and nitroaniline-modified silica gel, respectively. Comparison with other methods A critical review comparing several techniques for the measurement of cortisol levels in human plasma will be presented in a separate paper [ 151. In addition three sera were analyzed by HPLC and the eluted cortisol peaks were collected by a micro fraction-collector. The collected samples were analyzed by RIA [ 151 and the results were in very good agreement with the HPLC values.
TABLE
II
RECOVERY Solvent
OF
CORTISOL
AND Recovery (5%)
Ethyl Diethyl
PREDNISOLONE cortisol
FOR
Recovery (%)
acetate
89
82
ether
78
72
96
93
Dichloromethane
SOME
SOLVENTS prednisolone
169
Injected
amount
Cortisol
(ng)
Fig. 1. Calibration curves. o, cortisol test mixtures; a, cortisol extracted mixtures; ., prednisolone added to plasma and extracted from plasma.
from plasma;
0. pradnisolone
test
PREDNISOLONE
CORTISOL
10 TIMF
(min)
5
0
c--
Fig. 2. Chromatogram of plasma dichloromethanelpropan-2-al/water
sample. Column: 200 X 4.6 mm i.d.; Nucleosil NO2 ; dp = 5 pm. Eluent: (97.5 : 2.3 : 0.2. v/v); flow rate 1.15 ml/min. Detection: UV 240 nm.
A
10
20 + TIME (min)
B
C
u-LA_ )
10
20 TIME (min)
-
Fig. 3. Chromatogram of plasma sample: A. without addition of internal standard; B, with addition of internal standard. Column: 220 X 2.1 mm i.d.: modified LiCbrosorb SI 60: dp = 5 pm. Eluent: dichloromethane/propan-2-al/water (97.5 : 2.3 : 0.2, v/v); flow rate 25 ml/h. Detectibn: UV 240 nm. Sample: C. cortisol; P, prednisolone.
171
Fig. 4. Chromatogram
of a plasma sample of a patient
treat&d with dexsmathasone.
Conditions
see Fig. 3.
Positive identifications To show the selectivity of the liquid chromatographic method a chromatogram of a plasma of a patient treated with dexamethasone was made. Fig. 4 shows the result; the cortisol content is decreased drastically by this treatment to 2.5 E.cgcortisol/lOO ml plasma. Fig. 5 demonstrates the results that can be obtained by the off-line coupling of liquid chromatography and field desorption-mass spectrometry (FD-MS). The cortisol was collected as it eluted from the LC column and the wire emitter was submerged in the solution, containing lo-’ g cortisol/ml. The FD-MS of cortisol confirms the identification of the peak in the chromatogram of a plasma extract.
100
a0
no
=o
330
-
Fig. 5. Field desorption-mass a current
of 15 mA; source
spectrum temperature
of cortisol. 75’C.
sso
340
Equipment:
360
370
mb Varian Mat 711;
Emitter
10 f.fm heated
by
172
Acknowledgements We gratefully acknowledge the cooperation of Dr. E.B.M. de Jong, St. Elisabeth Hospital, Tilburg. Thanks are also due to P.J.M.W. Claassen, for technical assistance and Dr. J. van der Greef, University of Amsterdam, for performing FD-MS experiments. References 1
Huber,
J.F.K.,
Meijers,
2
Huber,
J.F.K.,
Alderlieste,
3
Fitzpatrick.
4
Henry,
R.A.,
5
Cavina.
G.,
F.A.
and
Metiers.
7
Hesse,
C..
C.A.M.,
8
Trefz,
F.K.,
Byrd.
9
Panis,
N.A.
(1974)
van den Brust,
O.E..
Berg, L.E.
S. (1973)
J.A.R.J. Hatzel.
and
D.J.
and
12
M&r,
13
Karger.
B.L.,
Martin,
14
Huber,
J.F.K.
(1975)
15
Thijssen.
Anal.
A.
Kochen.
Milley,
W. Sci.
J., Vonk.
I. and Halasz,
and Blanchard,
R.C.
M. and Z. Anal.
Aldercreutz.
J.F.
(1973)
Guiochon. Chem.
N.
J.F.K.
44.
111
Chem.
45,
1337
80.
(1972)
2.
Chem.
Anal.
Klin.
J. Chromatogr.
Sci.
9,
513
89 Chem.
Biochem.
107,
261, 12,
347
193
181
753 and Deelder,
I. (1973) Clin.
R.S.
(1977)
J. Chromatogr.
83,
Chem.
G. (1974) 277.
J. Chromatogr.
Z. Klin.
(1975)
Chem. Anal.
2310
J. Chromatogr.
Huber.
12,
Anal.
H. (1973)
45,
(1971)
(1973)
and
(1972)
Poppe,
Chem.
D. (1974)
J. Chromatogr.
J.H.M..
J.A.R.J.
H. and
and Die&man. Cantafora,
K.
Sebestian,
J.H.H..
Harren,
Hiilsman,
Pietrzik,
10
J.A. and
and Hulsman,
E.T..
Siggia.
Schmit, Moretti,
6
11
C.A.M.
19,
J. Chromatogr.
132.
421
15
710
Anal.
Chem.
46,
1640
341
H. and van den
Berg.
J.H.M.
(1977)
Clin.
Chim.
Acta,
submitted
