Clinical Toxicology
ISSN: 0009-9309 (Print) (Online) Journal homepage: http://www.tandfonline.com/loi/ictx18
Correlation of the “EMIT” with a Gas-Liquid Chromatographic Method for Determination of Antiepileptic Drugs in Plasma Arvind K. N. Nandedkar, Henn Kutt & Gordon F. Fairclough To cite this article: Arvind K. N. Nandedkar, Henn Kutt & Gordon F. Fairclough (1978) Correlation of the “EMIT” with a Gas-Liquid Chromatographic Method for Determination of Antiepileptic Drugs in Plasma, Clinical Toxicology, 12:4, 483-494, DOI: 10.3109/15563657809150018 To link to this article: http://dx.doi.org/10.3109/15563657809150018
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CLINICAL TOXICOLOGY 12(4), Pp. 483-494 (1978)
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Correlation of the “EMIT” with a Gas-Liquid Chromatographic Method for Determination of Antiepileptic Drugs in Plasma
ARVIND K. N. NANDEDKAR* Laboratory f o r Clinical Biochemistry The New York Hospital-Cornell Medical Center New York, New York 10021 H E ” KUTT Department of Neurology The New York Hospital-Cornell Medical Center New York, New York 10021 GORDON F. FAIRCLOUGH, JR. Laboratory f o r Clinical Biochemistry The New York Hospital-Cornell Medical Center New York, New York 10021
INTRODUCTION Close monitoring of antiepileptic d r u g blood levels h a s proven to be of value in the management of epileptic patients [l]. The s e a r c h f o r faster, m o r e convenient, and reliable methodology in reporting “close to the true” values of blood levels of the most commonly used *Address r e p r i n t requests to A.K.N.N 483 Copyright 0 1978 by Marcel Dekker, Inc. All Rights Reserved. Neither this work nor any part may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, microfilming. and recording, or by any information storage and retrieval system, without permission in writing from the publisher.
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NANDEDKAR, KUTT, AND FAIRCLOUGH
drugs, either as a principle drug o r in combination, has continued and resulted in the introduction of the "Enzyme Multiplied Immunoassay Technique" (EMIT) [2]. A few papers have already appeared comparing the EMIT system of assay for antiepileptic drugs with conventional gas-liquid chromatographic methods (GLC) [3-61. In a special issue of Clinical Chemistry, on "Monitoring Drugs in Biological Fluids," other comparative studies (high-performance liquid chromatography and adaption of the EMIT System on a centrifugal analyzer) for antiepileptic drug assay are also described by various authors [7]. Some of these publications have compared the total range of assayed values and reported an excellent correlation. We are reporting studies by the EMIT system compared with GLC and our observations on the statistical significance and correlation a t various levels of the antiepileptic drugs in patient specimens. MATERIALS AND METHODS Blood samples drawn in EDTA-containing Vacutainer tubes (lavend e r top, B/D Vacutainer) and submitted to the Neurology Department for routine anticonvulsant drug a s s a y s were chosen at random f o r the EMIT assay, All specimens submitted for such determinations are routinely assayed by GLC. After centrifugation the plasma was drawn, kept at 2-8°C in capped tubes, and comparative analyses were performed within 48 hr. The total period covered f o r this comparison was approximately 14 weeks. The EMIT reagents were purchased in kit form from SYVA Corporation, Palo Alto, California 94304. All calibrator standards and a control were also purchased from SYVA Corporation and were prepared according to instructions. The standard curve for each drug assay was drawn on the graph paper provided f o r the specific assay and for the specific lot number. An instruction booklet and a sufficient supply of graph papers are provided with each kit [8]. In the semi-automated system, a Gilford spectrophotometer (300-N) with a microcuvet (30"C-Thermocuvette, Model 3017 T ) and a S W A Timer-Printer (Model 2400) were connected and used according to the manufacturer's instructions. A SYVA Pipetter-Diluter (Carvo Scientific, Model 1500) to dispense 50 fl of the specimen and/or reagents A, B, and 250 p l of the buffer f o r dilution was used. The GLC analyses were performed according to the method of Kupferberg [ 9 ] , modified as follows: an additional internal standard (para-tolyl phenobarbital) was used t o calculate phenobarbital values, and samples were injected by an automatic sampler. The column conditions in GLC were as follows: column packing--% OV 17 on Gas Chrom Q (80-100 mesh); U-tube glass column, length 183 cm, ID 2 mm; oven temperature 210"C, isothermal; injection port temperature
ANTIEPILEPTIC DRUG ASSAY
485
270°C; FID te mper at ur e 300°C. A Hewlett Packard gas chromatograph (HP7610) equipped with an automatic HP liquid s a m p l e r (Model 7671A) and an integrating r ecor der (Model 3380A) was used. The calculations wer e based on peak areas of four calibrators which w ere run daily.
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R E S U L T S AND D I S C U S S I O N A set of data f or the day-to-day precision on 19 different days for all levels of the standard calibrators (SYVA Corporation) is presented i n Table 1. It can be seen that for a l l four drugs included in this study, the coefficient of variation was higher in the lower region of the stan-
TABLE 1. The Day-to-Day Precision of the EMITa Drug a s s ay
Level, pg/ml
SJ
S. D.
cv, 7%
Phenobarbital
5.0 10.0 20.0 40.0 80.0
62 101 146 181 218
2.4 6.8 4.2 4.1 4.3
3.8 6.7 2.9 2.2 1.9
Phenytoin
2. 5 5.0 10.0 20.0 30.0
44 60 79 104 116
3.3 3.9 2.9 4.4 3.4
7.5 6.6 3.6 4.2 2.9
P r i m idon e
2. 5 5.0 10.0 15.0 20.0
57 82 114 131 148
3.9 5.2 2.8 2.9 3.7
6.8 6.3 2. 5 2.3 2. 5
Carbamazepine
1.0 2. 5 5.0 7. 5 10.0
38 70 102 12 5 139
3.0 3.7 3.6 3.7 2.4
8.0 5.2 3.5 3.0 1.7
a
The average of about 19 separate determinations for each point on the curve, represented as AA-AAo. bThe numbers are rounded off to the near est whole number.
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486
NANDEDKAR, KUTT, AND FAIRCLOUGH
dard curve. A s the total curve is evaluated, the higher level of the drug concentration appears to approach minimal deviation. These variations a r e pertinent in t e r m s of o u r understanding of the standard c u r v e evaluation. It is observed that, at times, all points on the curve are not the function of a straight line. If the standard curve is divided into t h r e e levels (low, medium, and high) g r e a t e r coefficient of variation is observed a t the lower section of the curve, and g r e a t e r caution is to be exercised in the interpretation of lower levels of these drugs. A decision, therefore, has to be made by an experienced person as to the slope of the standard curve. The EMIT a s s a y s y s t e m has considerably improved since its initial introduction. The reagents a r e m o r e stable than e a r l i e r ones. The measurement time has been changed from 95 to 4 5 sec, while initial delay t i m e is unchanged. In o r d e r to conduct t h e a s s a y in the concentration mode instead of the absorbance mode, a concentration factor of 2.667 was introduced which provides fast and reproducible AA-AAo in 30 s e c . Statistically, the duplicate analyses of patient specimens gave satisfactory correlation, and the data a r e presented in Table 2. We still p r e f e r to include duplicate runs on each specimen as standard procedure to be included i n the methodology as a l r e a d y commented on by Pippenger [ l o ] , r a t h e r than rely on a single reading as suggested by Booker and Darcey [4]. Within-day reproducibility on 18 replicate analyses f o r phenobarbital, phenytoin, primidone, and carbamazepine (at levels confirmed by the gas-liquid chromatographic method f o r the patient specimens) is given in Table 3. The standard c u r v e p r e pared in the e a r l y p a r t of the day was used f o r calculating the drug concentration over a period of one working shift ( 8 hr). The control (SYVA Corp.) was analyzed in duplicate each t i m e a batch of specimens was analyzed. It appears that if the spectrophotometric settings are left "on" f o r the e n t i r e shift period ( 8 h r ) , it is usually unnecess a r y to run the e n t i r e standard c u r v e again that afternoon. This TABLE 2. Within-Day Reproducibility of Antiepileptic Drugs Drug
d m l
Mean, %
S. D.
C.V., %
Phenobarbital
(2 5. o)a
24.7
1.42
5.7
Phenytoin
( 12.0)
14.91
1.30
8.7
Primidone
( 10.0)
9.95
0.72
7.2
Carbamazepine
( 5.1)
4.98
0.25
5.0
aThe d r u g levels are given in parentheses. Initially the level f o r each d r u g was established by GLC. The results are calculated f o r 18 replicate analyses.
ANTIEPILEPTIC DRUG ASSAY
487
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TABLE 3. A Standard Statistical Correlation f o r EMIT Reagents between Duplicate Analyses of the Antiepileptic Drugs P r e s e n t in the Specimensa AED a s s a y
No. compared
Slope
Intercept
Correlation coefficient
Phenobarbital
81
0.99
0.80
0.99
Phenytoin
81
0.98
0.68
0.97
Primidone
27
0.99
0.79
0.96
Carbamaz epine
22
0. 98
0.78
0.94
aAll levels (pg/ml) of the drugs are included. amounts to considerable savings in reagent c o s t s i n c e each t i m e reagents A and B a r e utilized, approximately $1.75 is added to the cost of the test. The Gilford spectrophotometer (Model 300 N ) used in this study was stable over this period, and no f u r t h e r changes o r checks in initial calibrations w e r e required. Instructions f o r initial calibration of different types of spectrophotometers used in the EMIT a s s a y are provided with the kits by SYVA [8]. We a l s o confirmed the observation that m o r e consistent r e s u l t s w e r e obtained by waiting 1 min after the initial dilution before proceeding with the analysis [3]. The plasma specimens from many patients with a known history of intake of m o r e than one drug w e r e subjected to EMIT analysis. The concentrations of these drugs w e r e determined by o u r routine GLC procedure. A total of 179 patient specimens w e r e analyzed. The specimens showing values higher than the highest c a l i b r a t o r standard f o r EMIT a s s a y were diluted according to the SYVA instruction i n s e r t with each kit. No false positive o r false negative d r u g levels w e r e observed. Under the conditions of this study no c r o s s reactivity f o r the d r u g s apparently present in patient plasma could be detected. A computer-analyzed s e t of data f o r phenobarbital and phenytoin is provided in Table 4. F o r the control of s e i z u r e s , the majority of epileptics a r e p r e s c r i b e d either phenobarbital o r phenytoin, o r both of t h e s e drugs in combination. Therefore, r e s u l t s obtained f o r these d r u g s a t t h r e e different level ranges (low, medium, and high) by the EMIT and GLC are evaluated. The plasma levels (p.g/ml) w e r e divided on a n empirical b a s i s into: ( a ) low level (phenobarbital, 1-17; phenytoin, 2-10), ( b ) medium level (phenobarbital, 18-34; phenytoin, 10-20), and ( c ) high level (phenobarbital,>34; phenytoin, >20). In the phenobarbital a s s a y by EMIT, the maximum s p r e a d of values was observed in the high level; however, a better statistical correlation was observed i n this range than in low o r medium level ranges. In seven c a s e s of
00 03
A
pg/mlP
EMIT
Z
GLC
D. High values (> 34 r.lg/mljb
EMIT
2
GLC
(18-34
C. Medium values
EMIT
2
GLC
B. Low values (1- 17 pg/ml)a
EMIT
2
GLC
A. Total specimens (all v a l u e s p
I. Phenobarbital assay
24
24
-
34
34
-
22
22
-
80
-
80
Total no. of specimens
-
9
13
-
-
-
-
8
-
-
26
-
7
-
24 -
-
-
15
-
-
54
-
No. of No. of POS. neg.
53.20
50.77
-
25.58
24.20
-
12.2
11.79
-
30.19
-
28.76
Average
24.32
23.47
-
4.17
4.38
-
5.05
3.75
-
21.12
20.20
-
SD
Yes
-
Yes
Yes
-
Yes
Yes
-
Yes
Yes
-
Yes
0.797 -
Intercept
-
0.988
-
-
0.76
-
-
2.998
7.17 -
1.295 -3.067 -
-
1.022
-
Gaussian Slope
Regression line
-
0.945
-
0.799
0.962 -
-
0.977
Coefficient of correlations
TABLE 4. A Statistical Evaluation of the Phenobarbital and Phenytoin Assays by Gas Liquid Chromatography (GLC) and Enzyme Multiplied Immunoassay Technique (EMIT)
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W
03
rp
37
9 -
4 -
-
14
-
-
16
-
15
-
-
33
-
-
20 -
45 -
No. of No. of pos. neg.
The difference is significant. bThe difference is not significant.
a
14
EMIT
14
-
D. High values (> 20 pg/ml)a GLC
Z
30
30
-
37
EMIT
Z
GLC
C. Medium values (10-20 pg/ml)b
EMIT
-
B. Low values (2- 10 pg/ml)b GLC
Z
81
81
-
Total no. of specimens
GLC Z EMIT
A. Total specimens (all values ) b
a s sa y
II. Phenytoin
41.55
-
40.07
14.69
14.71
-
6.45
-
6. 53
15.57
15.36
-
Average
34.20
-
30.09
3.29
3.30
-
2.65
-
2.79
18.81
17.25
-
SD
0.817
Yes
-
Yes
Yes
-
Yes
-
5.626
-
-
0.726
-
-
1.124 -3.501 -
-
0.616
-
-
-
-
Yes
-
Intercept
1.077 -0.975
-
Yes
No
-
No
Gaussian Slope
Regression line
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0.989 -
-
0.619
-
-
0.922
-
0.988 -
Coefficient of correlations
1~9'
Section IIIe
Section I I ~
Section 13.8
29
11
40 37
9
10
11
42
9
8
4
28
7
13
21
6
3
11
5
34
22
4
2
10
3
15
5
1
17.8
24
2
18.3 19. 5
2.1 2.3 3.8 2.3 3.4 2.0 0.5
9.4 9. 5 14.0
7.7 13.8 12.8 1.2
11.5 11.8 17.8
1.7
14.8
17.2
10.0
17.9 0. 5
2.3
2.8
29.4
13. 5
19.8
23.0
21.3
18.7 1.4 6.1
9. 5
1.4 13.4
14.8
7. 5
13.7
9.5
6.8
0.9
-
-
-
A %
A (E-G)
43.0
8.6
19.2
20.3
30.5
16. 5
29.0
GLC ( G )
49.8
9.5
19.9
14
1 29.9
28.0
EMIT ( E )
8
Code no.
10
Specimen
TABLE 5. "Bottom of the Bottle Effect." A Comparison of the "EMIT" with GLC for the Assay of Phenytoina
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W
I&
X
r
0 c GI
F
*Z
z
0
19.0
19 43 32 16 31
7
8
9
10
11
33.0
5. 9 21.0
2.7
11.1
11.0
1.1
8. 5
6. 5
1.8
12.0
3.2
8.7
8.0
0.9
3.0
36.4
54.2
43.9
42.1
45.0
26.1
21.7
The values are expressed in pg/ml units. bAll the specimens and c a l i b r a t o r s a r e run in duplicate as a p a r t of the run. %ection I: The experiment was s t a r t e d with 3.5 m l of Reagent A and B. A standard curve consisted of c a l i b r a t o r s ( 1 negative and 5 with known concentrations of the drug). The Syva control and two specim e n s w e r e run in this section. A total of 1.1 m l of the Reagents A and B w e r e utilized. dSection XI: A total of 11 specimens were run in this section and from 2.4 ml of the Reagents A and B, 1.3 m l w e r e left f o r the Section In. eSection 111: In this section after 11 runs, the bottle was left with 0.2 m l of the Reagents A and B.
a
2.0
18
19.8
8.3 11. 5
21
5
6
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c
rp W
*>
% m
z
U
8
w r
__
$
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492
NANDEDKAR, KUTT, AND FAIRCLOUGH
patients known to be on this medication, the EMIT values would have led to an e r r o r in clinical judgment. This is confirmed by one of u s (Dr. Henn Kutt), who regularly attends the clinic. For phenobarbital, this constitutes about 9% of the patient population included in this study. Phenytoin overall provided a reasonably good correlation both statistically and clinically when assayed by both syst em s; and clinical judgment e r r o r s would have been few based on EMIT values alone. Primidone and carbamazepine al s o correlated well by both s y s tems and provided a satisfactory analytical and clinical picture. We would like to add a precautionary note because of our observations on the "Bottom of the Bottle Effect." It appeared that the EMIT values gradually approached higher numbers than GLC when only s ma ll amounts of reagents were left in the bottle, and this trend was observed in all of the assays. A systematic approach, as presented in Table 5, f o r the as s ay of phenytoin shows that the values gradually show a "climbing the ladder" effect when a small amount of reagent is left in the bottle. When repeated with newly prepared bottles of reagents, the values in these samples were considerably cl oser to the values obtained by GLC. A variance ratio test for the bottom of the bottle value is presented in Table 6, and statistically shows little cause f o r ala r m. However, the calculated F value in Section III is approximately 50% higher than in Section II. By the most conservative estimate, when reagent content is low in the bottle, the EMIT values may read at least 30% higher than GLC. In fact, these differences are particularly critical in the cas es of patients where fine titration is required to obtain a "maintenance dose" f or effective management of seizures. We suggest that as long as the s am e lot is available, by the time one approaches a level of approximately 0.7 ml of reagent in the bottle, another s e t of appropriate reagent should be added to the leftover. This can be judged by either keeping a record of the numbers on the p r in ter tape o r by preparing a marked bottle for this purpose. After thoroughly mixing the contents of the bottle, the controls and at least one e a r l i e r specimen should be checked before further analyses are performed. A s e t c r iter ia f or an appropriate value for an antiepileptic drug and the patient's clinical picture needs to be established by all concerned with the patient's care. The interfering sources in the EMIT system are s e v e r e hemolysis, lipemia, and severely jaundiced specimens [8]. The extent of e r r o r introduced by these factors may vary, and it may be difficult to decide what degree of lipemia o r hemolysis is enough to make a sample unsuitable for analyses by EMIT. F'urthermore, s o me c l e a r specimens do not duplicate well in the EMIT system. Presently, it appears that in such cas es GLC offers as a method of choice. A questionable result is always questionable; therefore, a rapport between the physician and the laboratorian is essential in o r d e r to minimize e r r o r in clinical judgment. This important concept was
ANTIEPILEPTIC DRUG ASSAY
493
TABLE 6. A Variance Ratio T es t ( F- T es t ) f o r the Assay of Phenytoin by "EMIT" and GLC: "Bottom of the Bottle EffectfTa ~~
Section II
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EMIT
Section lII
SD
Variance (SD') Fb
13.35
178.23
FC/ table
SD
Variance (SD') Fb
9.14
88.36
1.269 4.85 GLC
11.85
140.42
2.454
FC/ table
4.85
6.00 36.00
a
These data are derived from Table 5. b F = l a r g e r variance . s ma ller variance CF/table: F or P = 0.01, from E. S. Peterson and H. 0. Hartley (eds. ). Biometrica Tables f or Statisticians, 3rd ed., Cambridge University Press, Cambridge, London, 1966, Table 18. For degree of freedom (N-1) = 10. raised e a r l i e r in 1974 by Kutt and Penry [ l ] ,and a need for further study is still warranted. SUMMARY We report a study of the comparison of antiepileptic drugs (phenobarbital, phenytoin, primidone, and carbamazepine) by the Enzyme Multiplied Im munoassay Technique "EMIT" and the gas chromatography (GLC) method. Overall, reasonable correlations w ere observed f o r all of the above-mentioned as s ays by both methods in the majority of s amp les included in this study. Our observations f o r statistical and clinical differences at various levels of phenobarbital and phenytoin are discussed. A suggestion is provided in o r d e r to avoid a m aj or discrepancy (approximately 30% higher values by EMIT v s GLC) in res u lts observed from the "Bottom of the Bottle Effect" f o r EMIT reagent. ACKNOWLEDGMENTS We thank Ms. Karla Drgon for the excellent editing and typing of this manuscript. Miss Ann Detrich and Mr. B. Majumdar provided skillful technical as s i s t ance in the early phases of GLC analyses.
494
NANDEDKAR, KUTT, AND FAIRCLOUGH
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REFERENCES
H. Kutt and J. K. Penry, Usefulness of blood levels of antiepileptic drugs, Arch. Neurol., 31, 283 (1974). R. J. Bastiani, R. C. Philips, R. S. Schneider, and E. F. Ullman, Homogeneous immunochemical drug a s s a y s , Am. J. Med. Tech2 no1 , 39, 2 1 1 (1973). [31 M. Legas and V. A. Raisys, Correlation of the EMIT antiepilept i c drug a s s a y with a gas liquid chromatographic method, Clin. Biochem., 2, 35 (1976). [41 H. E. Booker and B. A. Darcey, Enzymatic immunoassay vs. g a s liquid chromatography f o r determination of phenobarbital and diphenylhydantoin in s e r u m , Clin. Chem., 21, 1766 (1975). [ 51 L. Sun and E. R Walwick, Primidone a n a l y s e s T C o r r e l a t i o n of g a s chromatographic a s s a y with enzyme immunoassay, Clin. Chem., - 22, 901 (1976). V. Spiechler, L. Sun, D. S. Miyada, S. G. Sarandis, E. R. Walwick, M. W. Klein, D. B. Jordan, and B. J e s s e n , Radioimmunoa s s a y , enzyme immunoassay, spectrophotometry and g a s liquid chromatography compared f o r determination of phenobarbital Clin. __ Chem., _ 22, 749 ( 1976). and diphenylhydantoin, _ Clinical Chemistry (J. S. King, ed. ), American Association f o r Clinical Chemistry, U.S., 22, ( 6 ) 1976. "EMIT-aed" Instruction Booklet 6A 164-2, S W A Corporation, P a l o Alto, California 94304. H. J. Kupferberg, Quantitative estimation of diphenylhydantoin, primidone and phenobarbital in plasma by gas-chromatography, Clin. Chem. Acta, 2 2 283 (1970). C. E. Pippenger, D. L. Sichler, and L. Lichtblau, P r e l i m i n a r y clinical evaluation of an experimental enzyme immunoassay s y s t e m f o r DPH and phenobarbital i n s e r u m , Clin. Chem., g, 869 (1974) Abstract.
ISSN: 0009-9309 (Print) (Online) Journal homepage: http://www.tandfonline.com/loi/ictx18
Correlation of the “EMIT” with a Gas-Liquid Chromatographic Method for Determination of Antiepileptic Drugs in Plasma Arvind K. N. Nandedkar, Henn Kutt & Gordon F. Fairclough To cite this article: Arvind K. N. Nandedkar, Henn Kutt & Gordon F. Fairclough (1978) Correlation of the “EMIT” with a Gas-Liquid Chromatographic Method for Determination of Antiepileptic Drugs in Plasma, Clinical Toxicology, 12:4, 483-494, DOI: 10.3109/15563657809150018 To link to this article: http://dx.doi.org/10.3109/15563657809150018
Published online: 25 Sep 2008.
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Date: 06 November 2015, At: 22:19
CLINICAL TOXICOLOGY 12(4), Pp. 483-494 (1978)
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Correlation of the “EMIT” with a Gas-Liquid Chromatographic Method for Determination of Antiepileptic Drugs in Plasma
ARVIND K. N. NANDEDKAR* Laboratory f o r Clinical Biochemistry The New York Hospital-Cornell Medical Center New York, New York 10021 H E ” KUTT Department of Neurology The New York Hospital-Cornell Medical Center New York, New York 10021 GORDON F. FAIRCLOUGH, JR. Laboratory f o r Clinical Biochemistry The New York Hospital-Cornell Medical Center New York, New York 10021
INTRODUCTION Close monitoring of antiepileptic d r u g blood levels h a s proven to be of value in the management of epileptic patients [l]. The s e a r c h f o r faster, m o r e convenient, and reliable methodology in reporting “close to the true” values of blood levels of the most commonly used *Address r e p r i n t requests to A.K.N.N 483 Copyright 0 1978 by Marcel Dekker, Inc. All Rights Reserved. Neither this work nor any part may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, microfilming. and recording, or by any information storage and retrieval system, without permission in writing from the publisher.
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484
NANDEDKAR, KUTT, AND FAIRCLOUGH
drugs, either as a principle drug o r in combination, has continued and resulted in the introduction of the "Enzyme Multiplied Immunoassay Technique" (EMIT) [2]. A few papers have already appeared comparing the EMIT system of assay for antiepileptic drugs with conventional gas-liquid chromatographic methods (GLC) [3-61. In a special issue of Clinical Chemistry, on "Monitoring Drugs in Biological Fluids," other comparative studies (high-performance liquid chromatography and adaption of the EMIT System on a centrifugal analyzer) for antiepileptic drug assay are also described by various authors [7]. Some of these publications have compared the total range of assayed values and reported an excellent correlation. We are reporting studies by the EMIT system compared with GLC and our observations on the statistical significance and correlation a t various levels of the antiepileptic drugs in patient specimens. MATERIALS AND METHODS Blood samples drawn in EDTA-containing Vacutainer tubes (lavend e r top, B/D Vacutainer) and submitted to the Neurology Department for routine anticonvulsant drug a s s a y s were chosen at random f o r the EMIT assay, All specimens submitted for such determinations are routinely assayed by GLC. After centrifugation the plasma was drawn, kept at 2-8°C in capped tubes, and comparative analyses were performed within 48 hr. The total period covered f o r this comparison was approximately 14 weeks. The EMIT reagents were purchased in kit form from SYVA Corporation, Palo Alto, California 94304. All calibrator standards and a control were also purchased from SYVA Corporation and were prepared according to instructions. The standard curve for each drug assay was drawn on the graph paper provided f o r the specific assay and for the specific lot number. An instruction booklet and a sufficient supply of graph papers are provided with each kit [8]. In the semi-automated system, a Gilford spectrophotometer (300-N) with a microcuvet (30"C-Thermocuvette, Model 3017 T ) and a S W A Timer-Printer (Model 2400) were connected and used according to the manufacturer's instructions. A SYVA Pipetter-Diluter (Carvo Scientific, Model 1500) to dispense 50 fl of the specimen and/or reagents A, B, and 250 p l of the buffer f o r dilution was used. The GLC analyses were performed according to the method of Kupferberg [ 9 ] , modified as follows: an additional internal standard (para-tolyl phenobarbital) was used t o calculate phenobarbital values, and samples were injected by an automatic sampler. The column conditions in GLC were as follows: column packing--% OV 17 on Gas Chrom Q (80-100 mesh); U-tube glass column, length 183 cm, ID 2 mm; oven temperature 210"C, isothermal; injection port temperature
ANTIEPILEPTIC DRUG ASSAY
485
270°C; FID te mper at ur e 300°C. A Hewlett Packard gas chromatograph (HP7610) equipped with an automatic HP liquid s a m p l e r (Model 7671A) and an integrating r ecor der (Model 3380A) was used. The calculations wer e based on peak areas of four calibrators which w ere run daily.
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R E S U L T S AND D I S C U S S I O N A set of data f or the day-to-day precision on 19 different days for all levels of the standard calibrators (SYVA Corporation) is presented i n Table 1. It can be seen that for a l l four drugs included in this study, the coefficient of variation was higher in the lower region of the stan-
TABLE 1. The Day-to-Day Precision of the EMITa Drug a s s ay
Level, pg/ml
SJ
S. D.
cv, 7%
Phenobarbital
5.0 10.0 20.0 40.0 80.0
62 101 146 181 218
2.4 6.8 4.2 4.1 4.3
3.8 6.7 2.9 2.2 1.9
Phenytoin
2. 5 5.0 10.0 20.0 30.0
44 60 79 104 116
3.3 3.9 2.9 4.4 3.4
7.5 6.6 3.6 4.2 2.9
P r i m idon e
2. 5 5.0 10.0 15.0 20.0
57 82 114 131 148
3.9 5.2 2.8 2.9 3.7
6.8 6.3 2. 5 2.3 2. 5
Carbamazepine
1.0 2. 5 5.0 7. 5 10.0
38 70 102 12 5 139
3.0 3.7 3.6 3.7 2.4
8.0 5.2 3.5 3.0 1.7
a
The average of about 19 separate determinations for each point on the curve, represented as AA-AAo. bThe numbers are rounded off to the near est whole number.
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486
NANDEDKAR, KUTT, AND FAIRCLOUGH
dard curve. A s the total curve is evaluated, the higher level of the drug concentration appears to approach minimal deviation. These variations a r e pertinent in t e r m s of o u r understanding of the standard c u r v e evaluation. It is observed that, at times, all points on the curve are not the function of a straight line. If the standard curve is divided into t h r e e levels (low, medium, and high) g r e a t e r coefficient of variation is observed a t the lower section of the curve, and g r e a t e r caution is to be exercised in the interpretation of lower levels of these drugs. A decision, therefore, has to be made by an experienced person as to the slope of the standard curve. The EMIT a s s a y s y s t e m has considerably improved since its initial introduction. The reagents a r e m o r e stable than e a r l i e r ones. The measurement time has been changed from 95 to 4 5 sec, while initial delay t i m e is unchanged. In o r d e r to conduct t h e a s s a y in the concentration mode instead of the absorbance mode, a concentration factor of 2.667 was introduced which provides fast and reproducible AA-AAo in 30 s e c . Statistically, the duplicate analyses of patient specimens gave satisfactory correlation, and the data a r e presented in Table 2. We still p r e f e r to include duplicate runs on each specimen as standard procedure to be included i n the methodology as a l r e a d y commented on by Pippenger [ l o ] , r a t h e r than rely on a single reading as suggested by Booker and Darcey [4]. Within-day reproducibility on 18 replicate analyses f o r phenobarbital, phenytoin, primidone, and carbamazepine (at levels confirmed by the gas-liquid chromatographic method f o r the patient specimens) is given in Table 3. The standard c u r v e p r e pared in the e a r l y p a r t of the day was used f o r calculating the drug concentration over a period of one working shift ( 8 hr). The control (SYVA Corp.) was analyzed in duplicate each t i m e a batch of specimens was analyzed. It appears that if the spectrophotometric settings are left "on" f o r the e n t i r e shift period ( 8 h r ) , it is usually unnecess a r y to run the e n t i r e standard c u r v e again that afternoon. This TABLE 2. Within-Day Reproducibility of Antiepileptic Drugs Drug
d m l
Mean, %
S. D.
C.V., %
Phenobarbital
(2 5. o)a
24.7
1.42
5.7
Phenytoin
( 12.0)
14.91
1.30
8.7
Primidone
( 10.0)
9.95
0.72
7.2
Carbamazepine
( 5.1)
4.98
0.25
5.0
aThe d r u g levels are given in parentheses. Initially the level f o r each d r u g was established by GLC. The results are calculated f o r 18 replicate analyses.
ANTIEPILEPTIC DRUG ASSAY
487
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TABLE 3. A Standard Statistical Correlation f o r EMIT Reagents between Duplicate Analyses of the Antiepileptic Drugs P r e s e n t in the Specimensa AED a s s a y
No. compared
Slope
Intercept
Correlation coefficient
Phenobarbital
81
0.99
0.80
0.99
Phenytoin
81
0.98
0.68
0.97
Primidone
27
0.99
0.79
0.96
Carbamaz epine
22
0. 98
0.78
0.94
aAll levels (pg/ml) of the drugs are included. amounts to considerable savings in reagent c o s t s i n c e each t i m e reagents A and B a r e utilized, approximately $1.75 is added to the cost of the test. The Gilford spectrophotometer (Model 300 N ) used in this study was stable over this period, and no f u r t h e r changes o r checks in initial calibrations w e r e required. Instructions f o r initial calibration of different types of spectrophotometers used in the EMIT a s s a y are provided with the kits by SYVA [8]. We a l s o confirmed the observation that m o r e consistent r e s u l t s w e r e obtained by waiting 1 min after the initial dilution before proceeding with the analysis [3]. The plasma specimens from many patients with a known history of intake of m o r e than one drug w e r e subjected to EMIT analysis. The concentrations of these drugs w e r e determined by o u r routine GLC procedure. A total of 179 patient specimens w e r e analyzed. The specimens showing values higher than the highest c a l i b r a t o r standard f o r EMIT a s s a y were diluted according to the SYVA instruction i n s e r t with each kit. No false positive o r false negative d r u g levels w e r e observed. Under the conditions of this study no c r o s s reactivity f o r the d r u g s apparently present in patient plasma could be detected. A computer-analyzed s e t of data f o r phenobarbital and phenytoin is provided in Table 4. F o r the control of s e i z u r e s , the majority of epileptics a r e p r e s c r i b e d either phenobarbital o r phenytoin, o r both of t h e s e drugs in combination. Therefore, r e s u l t s obtained f o r these d r u g s a t t h r e e different level ranges (low, medium, and high) by the EMIT and GLC are evaluated. The plasma levels (p.g/ml) w e r e divided on a n empirical b a s i s into: ( a ) low level (phenobarbital, 1-17; phenytoin, 2-10), ( b ) medium level (phenobarbital, 18-34; phenytoin, 10-20), and ( c ) high level (phenobarbital,>34; phenytoin, >20). In the phenobarbital a s s a y by EMIT, the maximum s p r e a d of values was observed in the high level; however, a better statistical correlation was observed i n this range than in low o r medium level ranges. In seven c a s e s of
00 03
A
pg/mlP
EMIT
Z
GLC
D. High values (> 34 r.lg/mljb
EMIT
2
GLC
(18-34
C. Medium values
EMIT
2
GLC
B. Low values (1- 17 pg/ml)a
EMIT
2
GLC
A. Total specimens (all v a l u e s p
I. Phenobarbital assay
24
24
-
34
34
-
22
22
-
80
-
80
Total no. of specimens
-
9
13
-
-
-
-
8
-
-
26
-
7
-
24 -
-
-
15
-
-
54
-
No. of No. of POS. neg.
53.20
50.77
-
25.58
24.20
-
12.2
11.79
-
30.19
-
28.76
Average
24.32
23.47
-
4.17
4.38
-
5.05
3.75
-
21.12
20.20
-
SD
Yes
-
Yes
Yes
-
Yes
Yes
-
Yes
Yes
-
Yes
0.797 -
Intercept
-
0.988
-
-
0.76
-
-
2.998
7.17 -
1.295 -3.067 -
-
1.022
-
Gaussian Slope
Regression line
-
0.945
-
0.799
0.962 -
-
0.977
Coefficient of correlations
TABLE 4. A Statistical Evaluation of the Phenobarbital and Phenytoin Assays by Gas Liquid Chromatography (GLC) and Enzyme Multiplied Immunoassay Technique (EMIT)
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W
03
rp
37
9 -
4 -
-
14
-
-
16
-
15
-
-
33
-
-
20 -
45 -
No. of No. of pos. neg.
The difference is significant. bThe difference is not significant.
a
14
EMIT
14
-
D. High values (> 20 pg/ml)a GLC
Z
30
30
-
37
EMIT
Z
GLC
C. Medium values (10-20 pg/ml)b
EMIT
-
B. Low values (2- 10 pg/ml)b GLC
Z
81
81
-
Total no. of specimens
GLC Z EMIT
A. Total specimens (all values ) b
a s sa y
II. Phenytoin
41.55
-
40.07
14.69
14.71
-
6.45
-
6. 53
15.57
15.36
-
Average
34.20
-
30.09
3.29
3.30
-
2.65
-
2.79
18.81
17.25
-
SD
0.817
Yes
-
Yes
Yes
-
Yes
-
5.626
-
-
0.726
-
-
1.124 -3.501 -
-
0.616
-
-
-
-
Yes
-
Intercept
1.077 -0.975
-
Yes
No
-
No
Gaussian Slope
Regression line
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0.989 -
-
0.619
-
-
0.922
-
0.988 -
Coefficient of correlations
1~9'
Section IIIe
Section I I ~
Section 13.8
29
11
40 37
9
10
11
42
9
8
4
28
7
13
21
6
3
11
5
34
22
4
2
10
3
15
5
1
17.8
24
2
18.3 19. 5
2.1 2.3 3.8 2.3 3.4 2.0 0.5
9.4 9. 5 14.0
7.7 13.8 12.8 1.2
11.5 11.8 17.8
1.7
14.8
17.2
10.0
17.9 0. 5
2.3
2.8
29.4
13. 5
19.8
23.0
21.3
18.7 1.4 6.1
9. 5
1.4 13.4
14.8
7. 5
13.7
9.5
6.8
0.9
-
-
-
A %
A (E-G)
43.0
8.6
19.2
20.3
30.5
16. 5
29.0
GLC ( G )
49.8
9.5
19.9
14
1 29.9
28.0
EMIT ( E )
8
Code no.
10
Specimen
TABLE 5. "Bottom of the Bottle Effect." A Comparison of the "EMIT" with GLC for the Assay of Phenytoina
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W
I&
X
r
0 c GI
F
*Z
z
0
19.0
19 43 32 16 31
7
8
9
10
11
33.0
5. 9 21.0
2.7
11.1
11.0
1.1
8. 5
6. 5
1.8
12.0
3.2
8.7
8.0
0.9
3.0
36.4
54.2
43.9
42.1
45.0
26.1
21.7
The values are expressed in pg/ml units. bAll the specimens and c a l i b r a t o r s a r e run in duplicate as a p a r t of the run. %ection I: The experiment was s t a r t e d with 3.5 m l of Reagent A and B. A standard curve consisted of c a l i b r a t o r s ( 1 negative and 5 with known concentrations of the drug). The Syva control and two specim e n s w e r e run in this section. A total of 1.1 m l of the Reagents A and B w e r e utilized. dSection XI: A total of 11 specimens were run in this section and from 2.4 ml of the Reagents A and B, 1.3 m l w e r e left f o r the Section In. eSection 111: In this section after 11 runs, the bottle was left with 0.2 m l of the Reagents A and B.
a
2.0
18
19.8
8.3 11. 5
21
5
6
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c
rp W
*>
% m
z
U
8
w r
__
$
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492
NANDEDKAR, KUTT, AND FAIRCLOUGH
patients known to be on this medication, the EMIT values would have led to an e r r o r in clinical judgment. This is confirmed by one of u s (Dr. Henn Kutt), who regularly attends the clinic. For phenobarbital, this constitutes about 9% of the patient population included in this study. Phenytoin overall provided a reasonably good correlation both statistically and clinically when assayed by both syst em s; and clinical judgment e r r o r s would have been few based on EMIT values alone. Primidone and carbamazepine al s o correlated well by both s y s tems and provided a satisfactory analytical and clinical picture. We would like to add a precautionary note because of our observations on the "Bottom of the Bottle Effect." It appeared that the EMIT values gradually approached higher numbers than GLC when only s ma ll amounts of reagents were left in the bottle, and this trend was observed in all of the assays. A systematic approach, as presented in Table 5, f o r the as s ay of phenytoin shows that the values gradually show a "climbing the ladder" effect when a small amount of reagent is left in the bottle. When repeated with newly prepared bottles of reagents, the values in these samples were considerably cl oser to the values obtained by GLC. A variance ratio test for the bottom of the bottle value is presented in Table 6, and statistically shows little cause f o r ala r m. However, the calculated F value in Section III is approximately 50% higher than in Section II. By the most conservative estimate, when reagent content is low in the bottle, the EMIT values may read at least 30% higher than GLC. In fact, these differences are particularly critical in the cas es of patients where fine titration is required to obtain a "maintenance dose" f or effective management of seizures. We suggest that as long as the s am e lot is available, by the time one approaches a level of approximately 0.7 ml of reagent in the bottle, another s e t of appropriate reagent should be added to the leftover. This can be judged by either keeping a record of the numbers on the p r in ter tape o r by preparing a marked bottle for this purpose. After thoroughly mixing the contents of the bottle, the controls and at least one e a r l i e r specimen should be checked before further analyses are performed. A s e t c r iter ia f or an appropriate value for an antiepileptic drug and the patient's clinical picture needs to be established by all concerned with the patient's care. The interfering sources in the EMIT system are s e v e r e hemolysis, lipemia, and severely jaundiced specimens [8]. The extent of e r r o r introduced by these factors may vary, and it may be difficult to decide what degree of lipemia o r hemolysis is enough to make a sample unsuitable for analyses by EMIT. F'urthermore, s o me c l e a r specimens do not duplicate well in the EMIT system. Presently, it appears that in such cas es GLC offers as a method of choice. A questionable result is always questionable; therefore, a rapport between the physician and the laboratorian is essential in o r d e r to minimize e r r o r in clinical judgment. This important concept was
ANTIEPILEPTIC DRUG ASSAY
493
TABLE 6. A Variance Ratio T es t ( F- T es t ) f o r the Assay of Phenytoin by "EMIT" and GLC: "Bottom of the Bottle EffectfTa ~~
Section II
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EMIT
Section lII
SD
Variance (SD') Fb
13.35
178.23
FC/ table
SD
Variance (SD') Fb
9.14
88.36
1.269 4.85 GLC
11.85
140.42
2.454
FC/ table
4.85
6.00 36.00
a
These data are derived from Table 5. b F = l a r g e r variance . s ma ller variance CF/table: F or P = 0.01, from E. S. Peterson and H. 0. Hartley (eds. ). Biometrica Tables f or Statisticians, 3rd ed., Cambridge University Press, Cambridge, London, 1966, Table 18. For degree of freedom (N-1) = 10. raised e a r l i e r in 1974 by Kutt and Penry [ l ] ,and a need for further study is still warranted. SUMMARY We report a study of the comparison of antiepileptic drugs (phenobarbital, phenytoin, primidone, and carbamazepine) by the Enzyme Multiplied Im munoassay Technique "EMIT" and the gas chromatography (GLC) method. Overall, reasonable correlations w ere observed f o r all of the above-mentioned as s ays by both methods in the majority of s amp les included in this study. Our observations f o r statistical and clinical differences at various levels of phenobarbital and phenytoin are discussed. A suggestion is provided in o r d e r to avoid a m aj or discrepancy (approximately 30% higher values by EMIT v s GLC) in res u lts observed from the "Bottom of the Bottle Effect" f o r EMIT reagent. ACKNOWLEDGMENTS We thank Ms. Karla Drgon for the excellent editing and typing of this manuscript. Miss Ann Detrich and Mr. B. Majumdar provided skillful technical as s i s t ance in the early phases of GLC analyses.
494
NANDEDKAR, KUTT, AND FAIRCLOUGH
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REFERENCES
H. Kutt and J. K. Penry, Usefulness of blood levels of antiepileptic drugs, Arch. Neurol., 31, 283 (1974). R. J. Bastiani, R. C. Philips, R. S. Schneider, and E. F. Ullman, Homogeneous immunochemical drug a s s a y s , Am. J. Med. Tech2 no1 , 39, 2 1 1 (1973). [31 M. Legas and V. A. Raisys, Correlation of the EMIT antiepilept i c drug a s s a y with a gas liquid chromatographic method, Clin. Biochem., 2, 35 (1976). [41 H. E. Booker and B. A. Darcey, Enzymatic immunoassay vs. g a s liquid chromatography f o r determination of phenobarbital and diphenylhydantoin in s e r u m , Clin. Chem., 21, 1766 (1975). [ 51 L. Sun and E. R Walwick, Primidone a n a l y s e s T C o r r e l a t i o n of g a s chromatographic a s s a y with enzyme immunoassay, Clin. Chem., - 22, 901 (1976). V. Spiechler, L. Sun, D. S. Miyada, S. G. Sarandis, E. R. Walwick, M. W. Klein, D. B. Jordan, and B. J e s s e n , Radioimmunoa s s a y , enzyme immunoassay, spectrophotometry and g a s liquid chromatography compared f o r determination of phenobarbital Clin. __ Chem., _ 22, 749 ( 1976). and diphenylhydantoin, _ Clinical Chemistry (J. S. King, ed. ), American Association f o r Clinical Chemistry, U.S., 22, ( 6 ) 1976. "EMIT-aed" Instruction Booklet 6A 164-2, S W A Corporation, P a l o Alto, California 94304. H. J. Kupferberg, Quantitative estimation of diphenylhydantoin, primidone and phenobarbital in plasma by gas-chromatography, Clin. Chem. Acta, 2 2 283 (1970). C. E. Pippenger, D. L. Sichler, and L. Lichtblau, P r e l i m i n a r y clinical evaluation of an experimental enzyme immunoassay s y s t e m f o r DPH and phenobarbital i n s e r u m , Clin. Chem., g, 869 (1974) Abstract.
