71
Clinica Chimica Acta, 78 (1977) 71-77 0 Elsevier/North-Holland Biomedical Press
CCA 8533
IMPROVED GAS CHROMATOGRAPHIC ANALYSIS CHLORPROMAZINE IN BLOOD SERUM
C.M. DAVIS Texas Research (U.S.A.) (Received
*, C.J. MEYER Institute
November
and D.C. FENIMORE
of Mental
lOth,
OF
Sciences,
Texas
Medical
Center,
Houston,
Texas
77030
1976)
Summary Reliable determinations of chlorpromazine levels in blood serum samples obtained from patients were accomplished by electron capture gas chromatography. By using modifications of the procedure to insure stability of the sample, minimal losses during sample preparation and gas chromatography, and by selecting appropriate operating parameters of the electron capture detector, excellent agreement was obtained in replicate analyses with a limit of sensitivity of 1 ng/ml in 1 ml of plasma.
Introduction A wide variation of chlorpromazine (CPZ) levels in blood serum has been reported both in the same patient and in comparisons between patients after receiving similar therapeutic dosages [ 1,2]. These observations together with evidence that schizophrenic patients significantly improve when the dosage is titrated to achieve defined blood levels [3] underscore the value of chlorpromazine assays as an aid to the clinician in determining drug dosage for the individual patient. Designing a suitable assay procedure, however, has not been without difficulty. The low blood concentrations of chlorpromazine encountered after therapeutic dosages, the complex metabolic pattern of the drug, and the many factors that hamper recovery of chlorpromazine from biological samples all contribute to the problem of devising reliable quantitative methods. Most clinical studies of chlorpromazine blood levels have employed the electron capture gas chromatographic (ECGC) procedure described by Curry [4] or modifications of this method [ 3-61, but a variety of problems are encountered when this analytical technique is applied to routine determinations, e.g. interference from other drugs or drug metabolites, variations in sample recovery, * To
whom
correspondence
should
be addressed.
72
and wide divergence of results in replicate determinations [ 71. Despite these disadvantages, ECGC yields levels of sensitivity that are not readily attainable with other methods but are necessary for the analysis. We have therefore incorporated further modifications in the ECGC procedure which improve significantly both the reproducibility and sensitivity of determinations of chlorpromazine blood levels in samples obtained from clinical subjects. The improvements involve primarily the use of promazine, a phenothiazine with very low electron affinity, as a “carrier” to increase recovery of chlorpromazine during the extractive and chromatographic processes. With loxapine as an internal standard, the chromatographic analysis employs a polar stationary phase (OV-225) as recommended by Whelpton and Curry [B] to improve separation. Using these techniques and other improvements in electron capture detection, determinations may be made with excellent reproducibility and accuracy and with a limit of detectability of 1 ng/ml on a serum volume of 1 ml. Procedure
Stock solutions Two stock solutions were prepared using 15% isoamyl alcohol/heptane as the solvent. The first contained chlorpromazine base at a concentration of 1 mg/ml and was used for preparing calibrations curves. The second contained promazine base (200 pg/ml) and loxapine * base (3.2 pg/ml). Small amounts of this solution were diluted 1 : 10 in heptane immediately before use for addition to serum samples. These stock solutions were stored in silylated amber bottles at 4°C or below and were stable over extended periods of time (6 months or more). All glassware was silylated by a vaporphase method [9] to reduce drug adsorption.
Preparation of serum samples A lOO-~1 amount of diluted stock solution
containing promazine, loxapine, and 1 ml of 1 M NaOH were added to 1 ml of serum in a silylated 15-ml disposable t.est tube having a teflon-lined screw cap. The serum was extracted with 10 ml of 1.5% isoamyl alcohol in heptane by gentle action on a tube rocker (Lab Industries, Berkeley, Calif.) for 30 min, followed by separation in a bench-top centrifuge for 5 min. The organic layer was transferred to another 15-ml tube and extracted with 1 ml of 0.05 M HCl by mixing on a vortex mixer and centrifuging. The organic layer was removed by aspiration and the aqueous phase alkalinized to pH 10 by the addition of 200 (~1of 1 M NH,OH. This portion, was then mixed with 2 ml of 1.5% isoamyl alcohol in heptane and centrifuged. The organic phase was transferred to a silylated Reacti-Vial (Pierce Chemical Co., Rockford, Ill.) and taken to dryness under reduced pressure. Extracted samples were chromatographed on the same day by dissolving the residue in 20 ~1 of 15% isoamyl alcohol in heptane, with care taken to contact the walls of the vial with the solvent. A 2-r.ll aliquot was injected onto the chromatographic column. * Z-Chloro-11-(4-methyl-l-piperaLinyl)-dibenz[b,f] was kindly
supplied
by Dr.
E.W.
Cantrall
[1,4]oxazepine; of
Lederle
Laboratories,
loxapine Pearl
River,
succinate; N.Y.
Loxitane@
73
Gas chromatography Gas chromatographic analyses were conducted with a 40 mCi 63Ni electron capture detector (ECD) operated with pulsed collection potential and analog linearization of detector signal [lo]. The pulse interval of the collection potential was adjusted for formation of maximum electron population [ 111, which, under the conditions of a well-conditioned column, purified carrier gas, and clean detector, was 1500 to 2000 psec. The gas chromatographic column was nickel-200 tubing (Handy and Harmon, Norristown, Pa.), 4 ft X l/S inch diameter, packed with 5% OV-225 on SO/l00 mesh Gas Chrom Q (Applied Science Laboratories, State College, Pa.). Column temperature was held at 220” C, injector and detector temperatures at 230°C and 31O”C, respectively, and the inlet of the column was arranged for “on-column ” injections. All connection fittings were brass which had been gold-plated (Atomex Immersion Gold, Engelhard Industries, Newark, N.J.). Carrier gas was zero-grade nitrogen which was routed through an Oxisorb gas purifier and a molecular sieve 5 A trap (Analabs, North Haven, Conn.) before entering the inlet system at 40 ml/min flow rate. Calibration Chlorpromazine concentration was determined by comparing the chlorpromazine-loxapine peak height ratio to known values displayed as a linear regression. Such calibration values may be obtained directly by chromatographic analysis of standard solutions. Errors may result, however, from differences in recovery of the compound in question and the internal standard when they are .WJ 24. .I% .10-
.M-
lo
50
KY)
ng/ml
SERA
r)40
Fig. 1. Calibration curve for chlorpromazine (CPZ,). The ordinate is the peak height ratio of chlorpromazine to the internal standard and the abscissa represents extractions of various concentrations of CPZ added to pooled sera.
74
extracted from serum. Therefore all calibration curves were obtained by adding known amounts of chlorpromazine to pooled, chlorpromazine-free serum, and determining values based on the complete analytical procedure. Fig. 1 shows a typical calibration curve with replicate determinations of known amounts of chlorpromazine ranging from 1.0 ng to 500 ng per ml. Routine linear regression analyses of the peak height ratios obtained in this manner yielded correlation coefficients greater than 0.999. Results and discussion Our initial attempts to reproduce published assay methods for chlorpromazine in blood serum at low ng/ml concentrations were for the most part unsuccessful. We found that the difficulties in the electron capture gas chromatographic procedure could be attributed primarily to instability of the blood samples, wide variability of extraction efficiency, introduction of interfering components during sample preparation, loss of chlorpromazine during extraction and chromatography, and errors associated with operation of the electron capture detector. Although this list of possible problems seems formidable, it is possible to attain satisfactory results by observing certain precautions. We shall consider each of these possible contributors to error, Sample stability Standard solutions of chlorpromazine in isoamyl alcohol/heptane were stable over long periods of time, particularly when they were stored in silylated amber glass bottles at 0°C or below. This stability was also observed in serum samples to which chlorpromazii~e had been added. Samples obtained from subjects to whom chlorpromazine had been administered, however, showed increasing amounts of chlorpromazine with time even when the samples were stored below 0°C. This elevation of chlorpromazine content may possibly result from reversion of a metabolite to the parent compound. For this reason the samples should be analyzed on the same day they are collected or, alternatively, the serum or serum extract should be stored at about -80°C. Comparison of sample extracts held at higher temperatures showed increases of as much as 100% in the chlorpromazine content over values obtained on the first day of sample collection. Extraction We investigated various methods for initial solvent extraction of CPZ from serum, such as rapid vortex mixing and shaking on mechanical devices or by hand;but we obtained the most satisfactory results from using a gentle rocking motion for 30 min. Emulsification was thus avoided, and extraction efficiencies were greater than 70% with a single 10 ml volume of extraction solvent. Extraction times shorter than 30 min contributed to divergent values in replicate determinations. Interference Although interference
the chromatographic process is capable of eliminating most of the encountered in analyses of complex mixtures, requisite resolution
75
is usually achieved at the expense of speed. Consequently, to avoid burdening the gas chromatographic separation, we tried to prevent introduction of extraneous materials during sample preparation. All solvents were therefore purified by fractional distillation and stored in bottles with teflon-sleeved glass stoppers. Screw-type plastic bottle caps, even those having teflon liners, proved to be a source of contamination which was difficult to separate from the CPZ peak on most of the chromatographic columns tested. Most interference from other drugs or drug metabolites that may be present in clinical samples can be eliminated by using OV-225 as the stationary phase in the chromatographic separation [ 81. A chromatogram of chlorpromazine extracted from blood serum is shown in Fig. 2. Of all the tranquilizers examined, including promazine, perphenazine, thioridazine, trifluoperazine, prochlorperazine, fluphenazine, haloperidol, chlordiazepoxide and diazepam, none interfered with CPZ using this procedure.
1
!
n
1
0
10
20
30
MINUTES Fig.
2. Typical
gas chromatograph
Fig.
3. Temperature
dependence
of a patient of electron
receiving capture
chlorpromazine
detector
response
therapy. to chlorpromazine.
76
Sample loss
A number of factors (incomplete extraction, adsorption to glassware surfaces, decomposition, adsorption to active sites in the chromatographic column) contribute to loss of chlorpromazine from the sample throughout the analytical procedure. The inclusion of an internal standard of similar chemical and physical properties to chlorpromazine helps to monitor these losses, and we found loxapine to be a convenient compound for this purpose, assuming, of course, that the blood serum samples are free of this drug. At very low levels of chlorpromazine, however, the internal standard did not compensate sufficiently for loss of the compound. When we added a relatively large amount of promazine to the blood serum to serve as a chlorpromazine carrier, the effect on recovery and reproducibility was significant. For example we compared two groups of seven serum samples, each containi~lg 10 ng/ml chlorpromazine and 25 ng/ml loxapine as the internal standard. One group contained 2 @g/ml of promazine, the other no added promazine. The sample set containing no carrier yielded a mean chlor~roma~ine/loxapi~~e peak height ratio of 0.065 and a standard deviation of 0.010. The group containing the promazine gave a mean ratio of 0.11 and a standard deviation of 0.0046. Electron capture detection Chlorpromazine reacts with thermal electrons by a dissociative electron capture process which like most Inonoch~orinated compounds, is highly energy dependent [ 121. The response of the electron capture detector to a constant concentration of chlorpromazine at varying detector temperatures is shown in Fig. 3. The steep slope of the curve illustrates the need to maintain constant electron energy during the detection process. This is difficult to achieve with a direct-current potential for eIectron collection where electron energy depends not only on the gas temperature in the detector but also on the strength of the electric field. To minimize contributions to electron energy other than thermal we used pulsed collection potentials [13], with pulse intervals in excess of 1000 psec for maximum detector sensitivity [ 111. To evaluate the accuracy and precision of the procedure, we analyzed fifteen blood serum samples to which 100 ng/ml of chlorpromazine was added. The mean value and standard deviation (S.D.) obtained from the series were 100.2 ng/ml and 0.070, respectively. Curry, however, has observed that values obtain, ed from in vitro determi~~ations may not be a reliable indicator of performance of the method when applied to analyses of in vivo samples [7]. Consequently, we examined fourteen blood samples from patients receiving chlorpromazine therapy by replicate determinations. Using the standard deviation value noted above as a percentage of the mean, all samples were within 95% confidence limits. Ten of the fourteen samples fell within one standard deviation of the mean with mean values ranging from 62.5 ngjml to 291 ng/ml. References 1
Curry,
S.H.,
Marshall.
S.H.,
Davis,
J.H.L..
Davis,
J.M.
and
Janowsky,
D.S.
(1970)
Arch.
Gem
Psychiatr.
22,
289-
(1970)
Arch
Gen.
Psvchiatr.
22.
209-
296 2
Curry, 215
J.W.,
JanowsIw,
D.S.
and
Marshall,
J.H.L.
77
3
Rivera-Calimfim,
4
Curry,
S.H.
5
Flint,
6
Christoph,
D.R.,
L.,
(1968)
Cast&&da, Anal.
Femllo, G.W.,
L. and Lasagna,
Chem.
C.F..
40,
Levandoski,
Schmidt,
D.E.,
L. (1973)
Clin.
Phmacol.
Ther.
14,
978-986
1251-1255 P. and
Davis,
Hwang,
J.M.
and
B. (1971)
Janowsky,
Clin. D.S.
Chem.
(1972)
17. Clin.
830-831 Chim.
Acta
38,
265-
270 ‘7 Curry. Usdin.
S.H. E..
(1974) eds.),
in The
pp.
8
Whelpton,
R.
9
Fenimore,
D.C..
and Curry, Davis,
10
Fenimore,
D.C.,
Zlatkis.
11
Fenimore,
D.C.
12
Wentworth,
13
Lovelock,
and
W.E., J.E.
Raven
S.H. C.M., A.
Davis,
Chen,
(1963)
Phenothiazines
335-345,
(1975)
J.H.
and Wentworth, C.M.
(19’70) 35.
Pharmacol.
and W.E.
J.E. 474
Related
Drugs
(Forrest,
I.S..
C~II,
C.rJ. and
York 27,
Harrington, (1968)
J. Chromatogr.
Lovelock,
Chem.
New
J. Pharm.
Whitford.
E. and
Anal.
and Structurally
Press,
(1966)
970
C.A. Anal.
Sci.
(1976)
Chem.
Anal. 40.
8. 519-523
J. Phys.
Chem.
70,
Chem.
1594-1596 445
48,
2289-2290
Clinica Chimica Acta, 78 (1977) 71-77 0 Elsevier/North-Holland Biomedical Press
CCA 8533
IMPROVED GAS CHROMATOGRAPHIC ANALYSIS CHLORPROMAZINE IN BLOOD SERUM
C.M. DAVIS Texas Research (U.S.A.) (Received
*, C.J. MEYER Institute
November
and D.C. FENIMORE
of Mental
lOth,
OF
Sciences,
Texas
Medical
Center,
Houston,
Texas
77030
1976)
Summary Reliable determinations of chlorpromazine levels in blood serum samples obtained from patients were accomplished by electron capture gas chromatography. By using modifications of the procedure to insure stability of the sample, minimal losses during sample preparation and gas chromatography, and by selecting appropriate operating parameters of the electron capture detector, excellent agreement was obtained in replicate analyses with a limit of sensitivity of 1 ng/ml in 1 ml of plasma.
Introduction A wide variation of chlorpromazine (CPZ) levels in blood serum has been reported both in the same patient and in comparisons between patients after receiving similar therapeutic dosages [ 1,2]. These observations together with evidence that schizophrenic patients significantly improve when the dosage is titrated to achieve defined blood levels [3] underscore the value of chlorpromazine assays as an aid to the clinician in determining drug dosage for the individual patient. Designing a suitable assay procedure, however, has not been without difficulty. The low blood concentrations of chlorpromazine encountered after therapeutic dosages, the complex metabolic pattern of the drug, and the many factors that hamper recovery of chlorpromazine from biological samples all contribute to the problem of devising reliable quantitative methods. Most clinical studies of chlorpromazine blood levels have employed the electron capture gas chromatographic (ECGC) procedure described by Curry [4] or modifications of this method [ 3-61, but a variety of problems are encountered when this analytical technique is applied to routine determinations, e.g. interference from other drugs or drug metabolites, variations in sample recovery, * To
whom
correspondence
should
be addressed.
72
and wide divergence of results in replicate determinations [ 71. Despite these disadvantages, ECGC yields levels of sensitivity that are not readily attainable with other methods but are necessary for the analysis. We have therefore incorporated further modifications in the ECGC procedure which improve significantly both the reproducibility and sensitivity of determinations of chlorpromazine blood levels in samples obtained from clinical subjects. The improvements involve primarily the use of promazine, a phenothiazine with very low electron affinity, as a “carrier” to increase recovery of chlorpromazine during the extractive and chromatographic processes. With loxapine as an internal standard, the chromatographic analysis employs a polar stationary phase (OV-225) as recommended by Whelpton and Curry [B] to improve separation. Using these techniques and other improvements in electron capture detection, determinations may be made with excellent reproducibility and accuracy and with a limit of detectability of 1 ng/ml on a serum volume of 1 ml. Procedure
Stock solutions Two stock solutions were prepared using 15% isoamyl alcohol/heptane as the solvent. The first contained chlorpromazine base at a concentration of 1 mg/ml and was used for preparing calibrations curves. The second contained promazine base (200 pg/ml) and loxapine * base (3.2 pg/ml). Small amounts of this solution were diluted 1 : 10 in heptane immediately before use for addition to serum samples. These stock solutions were stored in silylated amber bottles at 4°C or below and were stable over extended periods of time (6 months or more). All glassware was silylated by a vaporphase method [9] to reduce drug adsorption.
Preparation of serum samples A lOO-~1 amount of diluted stock solution
containing promazine, loxapine, and 1 ml of 1 M NaOH were added to 1 ml of serum in a silylated 15-ml disposable t.est tube having a teflon-lined screw cap. The serum was extracted with 10 ml of 1.5% isoamyl alcohol in heptane by gentle action on a tube rocker (Lab Industries, Berkeley, Calif.) for 30 min, followed by separation in a bench-top centrifuge for 5 min. The organic layer was transferred to another 15-ml tube and extracted with 1 ml of 0.05 M HCl by mixing on a vortex mixer and centrifuging. The organic layer was removed by aspiration and the aqueous phase alkalinized to pH 10 by the addition of 200 (~1of 1 M NH,OH. This portion, was then mixed with 2 ml of 1.5% isoamyl alcohol in heptane and centrifuged. The organic phase was transferred to a silylated Reacti-Vial (Pierce Chemical Co., Rockford, Ill.) and taken to dryness under reduced pressure. Extracted samples were chromatographed on the same day by dissolving the residue in 20 ~1 of 15% isoamyl alcohol in heptane, with care taken to contact the walls of the vial with the solvent. A 2-r.ll aliquot was injected onto the chromatographic column. * Z-Chloro-11-(4-methyl-l-piperaLinyl)-dibenz[b,f] was kindly
supplied
by Dr.
E.W.
Cantrall
[1,4]oxazepine; of
Lederle
Laboratories,
loxapine Pearl
River,
succinate; N.Y.
Loxitane@
73
Gas chromatography Gas chromatographic analyses were conducted with a 40 mCi 63Ni electron capture detector (ECD) operated with pulsed collection potential and analog linearization of detector signal [lo]. The pulse interval of the collection potential was adjusted for formation of maximum electron population [ 111, which, under the conditions of a well-conditioned column, purified carrier gas, and clean detector, was 1500 to 2000 psec. The gas chromatographic column was nickel-200 tubing (Handy and Harmon, Norristown, Pa.), 4 ft X l/S inch diameter, packed with 5% OV-225 on SO/l00 mesh Gas Chrom Q (Applied Science Laboratories, State College, Pa.). Column temperature was held at 220” C, injector and detector temperatures at 230°C and 31O”C, respectively, and the inlet of the column was arranged for “on-column ” injections. All connection fittings were brass which had been gold-plated (Atomex Immersion Gold, Engelhard Industries, Newark, N.J.). Carrier gas was zero-grade nitrogen which was routed through an Oxisorb gas purifier and a molecular sieve 5 A trap (Analabs, North Haven, Conn.) before entering the inlet system at 40 ml/min flow rate. Calibration Chlorpromazine concentration was determined by comparing the chlorpromazine-loxapine peak height ratio to known values displayed as a linear regression. Such calibration values may be obtained directly by chromatographic analysis of standard solutions. Errors may result, however, from differences in recovery of the compound in question and the internal standard when they are .WJ 24. .I% .10-
.M-
lo
50
KY)
ng/ml
SERA
r)40
Fig. 1. Calibration curve for chlorpromazine (CPZ,). The ordinate is the peak height ratio of chlorpromazine to the internal standard and the abscissa represents extractions of various concentrations of CPZ added to pooled sera.
74
extracted from serum. Therefore all calibration curves were obtained by adding known amounts of chlorpromazine to pooled, chlorpromazine-free serum, and determining values based on the complete analytical procedure. Fig. 1 shows a typical calibration curve with replicate determinations of known amounts of chlorpromazine ranging from 1.0 ng to 500 ng per ml. Routine linear regression analyses of the peak height ratios obtained in this manner yielded correlation coefficients greater than 0.999. Results and discussion Our initial attempts to reproduce published assay methods for chlorpromazine in blood serum at low ng/ml concentrations were for the most part unsuccessful. We found that the difficulties in the electron capture gas chromatographic procedure could be attributed primarily to instability of the blood samples, wide variability of extraction efficiency, introduction of interfering components during sample preparation, loss of chlorpromazine during extraction and chromatography, and errors associated with operation of the electron capture detector. Although this list of possible problems seems formidable, it is possible to attain satisfactory results by observing certain precautions. We shall consider each of these possible contributors to error, Sample stability Standard solutions of chlorpromazine in isoamyl alcohol/heptane were stable over long periods of time, particularly when they were stored in silylated amber glass bottles at 0°C or below. This stability was also observed in serum samples to which chlorpromazii~e had been added. Samples obtained from subjects to whom chlorpromazine had been administered, however, showed increasing amounts of chlorpromazine with time even when the samples were stored below 0°C. This elevation of chlorpromazine content may possibly result from reversion of a metabolite to the parent compound. For this reason the samples should be analyzed on the same day they are collected or, alternatively, the serum or serum extract should be stored at about -80°C. Comparison of sample extracts held at higher temperatures showed increases of as much as 100% in the chlorpromazine content over values obtained on the first day of sample collection. Extraction We investigated various methods for initial solvent extraction of CPZ from serum, such as rapid vortex mixing and shaking on mechanical devices or by hand;but we obtained the most satisfactory results from using a gentle rocking motion for 30 min. Emulsification was thus avoided, and extraction efficiencies were greater than 70% with a single 10 ml volume of extraction solvent. Extraction times shorter than 30 min contributed to divergent values in replicate determinations. Interference Although interference
the chromatographic process is capable of eliminating most of the encountered in analyses of complex mixtures, requisite resolution
75
is usually achieved at the expense of speed. Consequently, to avoid burdening the gas chromatographic separation, we tried to prevent introduction of extraneous materials during sample preparation. All solvents were therefore purified by fractional distillation and stored in bottles with teflon-sleeved glass stoppers. Screw-type plastic bottle caps, even those having teflon liners, proved to be a source of contamination which was difficult to separate from the CPZ peak on most of the chromatographic columns tested. Most interference from other drugs or drug metabolites that may be present in clinical samples can be eliminated by using OV-225 as the stationary phase in the chromatographic separation [ 81. A chromatogram of chlorpromazine extracted from blood serum is shown in Fig. 2. Of all the tranquilizers examined, including promazine, perphenazine, thioridazine, trifluoperazine, prochlorperazine, fluphenazine, haloperidol, chlordiazepoxide and diazepam, none interfered with CPZ using this procedure.
1
!
n
1
0
10
20
30
MINUTES Fig.
2. Typical
gas chromatograph
Fig.
3. Temperature
dependence
of a patient of electron
receiving capture
chlorpromazine
detector
response
therapy. to chlorpromazine.
76
Sample loss
A number of factors (incomplete extraction, adsorption to glassware surfaces, decomposition, adsorption to active sites in the chromatographic column) contribute to loss of chlorpromazine from the sample throughout the analytical procedure. The inclusion of an internal standard of similar chemical and physical properties to chlorpromazine helps to monitor these losses, and we found loxapine to be a convenient compound for this purpose, assuming, of course, that the blood serum samples are free of this drug. At very low levels of chlorpromazine, however, the internal standard did not compensate sufficiently for loss of the compound. When we added a relatively large amount of promazine to the blood serum to serve as a chlorpromazine carrier, the effect on recovery and reproducibility was significant. For example we compared two groups of seven serum samples, each containi~lg 10 ng/ml chlorpromazine and 25 ng/ml loxapine as the internal standard. One group contained 2 @g/ml of promazine, the other no added promazine. The sample set containing no carrier yielded a mean chlor~roma~ine/loxapi~~e peak height ratio of 0.065 and a standard deviation of 0.010. The group containing the promazine gave a mean ratio of 0.11 and a standard deviation of 0.0046. Electron capture detection Chlorpromazine reacts with thermal electrons by a dissociative electron capture process which like most Inonoch~orinated compounds, is highly energy dependent [ 121. The response of the electron capture detector to a constant concentration of chlorpromazine at varying detector temperatures is shown in Fig. 3. The steep slope of the curve illustrates the need to maintain constant electron energy during the detection process. This is difficult to achieve with a direct-current potential for eIectron collection where electron energy depends not only on the gas temperature in the detector but also on the strength of the electric field. To minimize contributions to electron energy other than thermal we used pulsed collection potentials [13], with pulse intervals in excess of 1000 psec for maximum detector sensitivity [ 111. To evaluate the accuracy and precision of the procedure, we analyzed fifteen blood serum samples to which 100 ng/ml of chlorpromazine was added. The mean value and standard deviation (S.D.) obtained from the series were 100.2 ng/ml and 0.070, respectively. Curry, however, has observed that values obtain, ed from in vitro determi~~ations may not be a reliable indicator of performance of the method when applied to analyses of in vivo samples [7]. Consequently, we examined fourteen blood samples from patients receiving chlorpromazine therapy by replicate determinations. Using the standard deviation value noted above as a percentage of the mean, all samples were within 95% confidence limits. Ten of the fourteen samples fell within one standard deviation of the mean with mean values ranging from 62.5 ngjml to 291 ng/ml. References 1
Curry,
S.H.,
Marshall.
S.H.,
Davis,
J.H.L..
Davis,
J.M.
and
Janowsky,
D.S.
(1970)
Arch.
Gem
Psychiatr.
22,
289-
(1970)
Arch
Gen.
Psvchiatr.
22.
209-
296 2
Curry, 215
J.W.,
JanowsIw,
D.S.
and
Marshall,
J.H.L.
77
3
Rivera-Calimfim,
4
Curry,
S.H.
5
Flint,
6
Christoph,
D.R.,
L.,
(1968)
Cast&&da, Anal.
Femllo, G.W.,
L. and Lasagna,
Chem.
C.F..
40,
Levandoski,
Schmidt,
D.E.,
L. (1973)
Clin.
Phmacol.
Ther.
14,
978-986
1251-1255 P. and
Davis,
Hwang,
J.M.
and
B. (1971)
Janowsky,
Clin. D.S.
Chem.
(1972)
17. Clin.
830-831 Chim.
Acta
38,
265-
270 ‘7 Curry. Usdin.
S.H. E..
(1974) eds.),
in The
pp.
8
Whelpton,
R.
9
Fenimore,
D.C..
and Curry, Davis,
10
Fenimore,
D.C.,
Zlatkis.
11
Fenimore,
D.C.
12
Wentworth,
13
Lovelock,
and
W.E., J.E.
Raven
S.H. C.M., A.
Davis,
Chen,
(1963)
Phenothiazines
335-345,
(1975)
J.H.
and Wentworth, C.M.
(19’70) 35.
Pharmacol.
and W.E.
J.E. 474
Related
Drugs
(Forrest,
I.S..
C~II,
C.rJ. and
York 27,
Harrington, (1968)
J. Chromatogr.
Lovelock,
Chem.
New
J. Pharm.
Whitford.
E. and
Anal.
and Structurally
Press,
(1966)
970
C.A. Anal.
Sci.
(1976)
Chem.
Anal. 40.
8. 519-523
J. Phys.
Chem.
70,
Chem.
1594-1596 445
48,
2289-2290
