Enzyme-linked immunosorbent assay for amitriptyline and other antidepressants using a monoclonal antibody

257

Clmicu Chimlcu Aria, 159 (1986) 251-261 Elsevier

CCA 03582

Enzyme-linked immunosorbent assay for amitriptyline and other antidepressants using a monoclonal antibody Helene

Denis,

Stefano

Marullo,

Johan

Hoebeke

and A. Donny

Strosberg

L.uhoruror):o/Molecular Immunolo~, Instrtut Jacques Monad, CNRS and Unwers~t,v Pans VII. 2 Place Jussieu, F 75251 Purrs cedex 05 (France) (Received

19 January

Key words: Amitnptyline;

1986; revision 21 March

Enzyme immunoassay;

1986)

Monoclonal antibodv

Summary We describe and evaluate a method to measure amitriptyline and other tricyclic antidepressants by enzyme linked immunosorbent assay, using monoclonal antibody. In this assay, biological samples were first incubated with the antibody; in a second step, free remaining antibody was allowed to bind to lysozyme-nortriptyline coated immunotitration plates. The bound fraction of the monoclonal antibody was revealed with rabbit anti-mouse serum coupled to horseradish peroxidase. The optical density of the reaction product was measured with a calorimeter at 410 nm. Specificity of the antibody was investigated by means of a Farr test showing interferences in therapeutic ranges only for chlorpromazine and phenytoine. Means of intra- and inter-assay variations were 10 and 13%, respectively. The results when compared to those obtained by gas chromatography with a selective nitrogen detector gave a correlation coefficient of 0.897. Finally, the great reliability of the monoclonal antibody, the advantages of a decreased analysis time, low cost and high capacity of the procedure contribute to make this immunoassay most suitable for clinical monitoring and pharmacokinetic studies of tricyclic antidepressants.

Introduction The treatment of depression commonly involves tricyclic antidepressants such as amitriptyline and imipramine. Many reports stress the difficulty of providing patients with effective dosage, because of the great variability of tricyclic antidepressants’ metabolism among patients which does not allow the derermination of a general relation between dose, plasma level and clinical response [l--4]. Neverthe0009~8981/86/$03.50

0 1986 Elsevier Science Publishers

B.V. (Biomedical

Division)

less. it is essential to establish the individual therapeutic zone in \vhlch 3 ,\tcacf clinical status is achieved [I] without risk of overdose. Such int[~xicati~~ns. leading t(t cardiac and neurologic toxicity and eventually to death 15.61, should he promptly diagnosed to decrease morbidity. Relationships between clinical response and plasma concentration have long been studied for amitriptyline, imipramine and their active metabolites: nortriptyline and desmethylimipramine. As their relative effects could not be clearfy distinguished. many authors suggested a correlation between clinical observations and the total concentration of both amitriptyline and nortriptyline. or imipramine and desmethylimipramine. Several techniques have already been proposed for measuring the concentrations of these drugs [7]: gas chromatography (GC) [X], high-pressure liquid chromatography (HPLC), spectrofluorimetry. and, more recently. radioimmunoassay (RIA) [9-141, and immunoenzymatic assays [ 151. Although their specificity. precision, and accuracy place GC, HPLC and RIA as reference techniques, feh medical services and laboratories can afford to use them for routine purpose. GC and HPLC have a high cost and need specially trained technicians while RIA implies the use of radiolabeled compounds which is restricted in many countries. This explains the need for the development of easier and less costly immunoenzymatic tests [16]. In this report. we describe an enzyme-linked immunosorbent assay (ELISA) using a single monoclonal antibody (mah) for the detection of tricyclic antidepressants in clinical monitoring.

Materiats and methods

Appurutus Dynatech

ELISA

Minireader,

and Dynatech

Immulon

plates (Fresnes,

France).

didesipramine and oxaprotiline were lmipramine, desipramine, clomipramine, kind gifts from Ciba-Geigy Laboratories (Basel, Switzerland), protriptyline from Merck-Sharp-Dohme, carbamazepine from Rhone-Poulenc (Vitry, France), amitriptyline, chlordi~epoxide, diazepam and nitrazepam from Roche (Neuillysur-Seine, France), 2-OH imipramine and 2-OH desipramine from Syntex Lahoratories (Palo Alto, CA, USA), doxepine and maprotiline from Prof. Agid, Pitie Salpetriere Hospital (Paris, France), promethazine, levomepromazine, chlorpromafrom Squibb zine and prochlorperazine from Specia (Paris, France). flufenazine (Neuilly-sur-Seine, France), phenytoine from Carrion (Nanterre, France), droperidol and sulpiride from Deand haloperidol from Janssen Lebrun (Paris, France), lagrange Laboratories (Chilly-Mazarin, France). Tween-20, bovine serum albumin (BSA) fraction V, lysozyme and azino-di(3ethyl-benzothiazoline-sulfonic acid) (ABTS) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Tritiated desipramine (‘H-DMI) was purchased from New England Nuclear (Boston. MA, USA).

259

Reagents Sodium phosphate buffer 0.01 mol/l in NaCl 0.15 mol/l, pH 7.4 (PBS), and PBS-gelatin-tween (PGT) containing 2.5 g of gelatin and tween-20 (1 ml/l). PBSglycine HCl solution: 0.2 mol/l glycine in PBS, adjusted to pH 2.5 with HCl. Diluted by half in PBS when coating the plates. The lysozyme-nortriptyline conjugate was prepared as previously described [18], following the procedure used by Kamel et al [17] for coupling the nortriptyline to the BSA; the coating solution contained 15 mg/ml lysozyme with 5 X lop3 mol/l nortriptyline, and was diluted to 10 pg/ml of conjugate in PBS-glycine-HCl buffer for coating ELISA plates. Amitriptyline stock solution at 2 x 10e3 mol/l was dissolved in 50 ~1 ethanol and then in water. Rabbit anti-mouse IgG antiserum, conjugated to peroxidase (RAM Pox) was purchased from Miles Chemicals (Naperville, IL, USA) and was used diluted 800-fold with PGT. ABTS: this substrate of the peroxidase was used at a concentration of 3.5 mmol/l in buffer (0.1 mol/l sodium acetate, 0.05 mol/l sodium phosphate NaH,PO,), H,O, (100 ~1 of 1% v/v in 10 ml of diluted substrate). Optical density of the green reaction product, was measured on the Dynatech ELISA Minireader at 410 nm. The monoclonal ANT antibody: this monoclonal antibody (mab), directed against nortriptyline, was prepared and characterized in our laboratory [18]. ANT mab was amplified as ascites in BALB/c female mice, which were primed with Pristane, then injected intraperitoneally with the mab producing hybridoma. At least 10 days later, ascites were collected, centrifuged to remove cells (10 min, at 4°C 5630 X g), and supernatant was kept at -20°C for future purification and use. Ascites supernatant was tested by electrophoresis on cellulose acetate strips (25 min migration, in sodium barbitone buffer 0.1 mol/l, pH 8.6, Shandon Southern Products, England; Rouge Ponceau as staining reagent 5 min; 5% v/v acetic acid as successive destaining baths) to evaluate the mab concentration. Purification of the ANT mab was performed by affinity chromatography on a Sepharose-nortriptyline column, prepared as previously described [18]. The same purified stock solution was used for all the assays. Determination of binding properties of ANT mab Binding properties of the mab were determined by means of the Farr technique [20] as previously described [18]. Briefly, the Ki for tested drugs were calculated from IC,, values determined with inhibition binding experiments using 2 X lo-’ mol/l 3H-DMI as labeled ligand. Inhibition binding experiments were performed in PBS pH 7.4 at 21°C and separation of bound ligand from free was obtained by precipitation at 60% (NH,),SO, followed by filtration on GF/F Whatman glass filters. Radioactivity was measured in a liquid scintillation counter after addition of 1 ml of Biofluor (New England Nuclear). IC,, were determined from at least 3 independent experiments.

Samples were gathered from Fernand Widal Hospital (Paris. France) I’ronl patients undergoing long-term therapy or patients intoxicated with tricyclic antidepressant. Blood was collected into heparinized tubes. avoiding Vacutainera and their rubber stoppers which reduce the drug concentration [7.19]. Centrifuged (IO min. at 4°C. 5630 X g) as soon as possible. samples were stored at 4°C and tested by Gas Chromatography with a selective nitrogen detector following the method described by Bailey and Jatlow [8]. Immediately afterwards. residual samples were frozen and kept at -20°C for analysis by the ELISA method. Drug-free blood was also collected and pooled for the calibration curve. It should be noted that during the first months of the investigation of this technique. a few aberrant low results were obtained. This was due to peroxidase activity of contaminating bacteria or hemolyzed samples which resulted in maximum ahsorbances and thus no detectable inhibition. This non-specific interference was avoided by: (i) rapid storage at -20°C after collection and separation of plasma. (ii) discarding of hemolyzed samples, (iii) centrifugation of precipitating samples. Patient samples were tested without dilution when an ordinary treatment check-up was requested, or diluted 2- to IO-fold in drug-free plasma when a high concentration or an overdose was suspected. For intra- and interassay procedures. plasma samples and calibration curve drug-free plasma were split into portions and stored at -20°C.

ELISA procedure Determination of coating antigen and mab dilutions to be used in ELISA, was by a ‘two dimensional’ test. In this test both LNT and mab were used at different concentrations, and conditions were chosen that gave 50% of maximal absorbance obtained by the immunoperoxidase reaction. In the assays. final dilutions of LNT and purified mab were respectively 1 : 1500 and 1 : 200. On the first day of the assay. patients’ samples, standards and controls were incubated overnight at 4°C with ANT mab; 100 ~1 of plasma. standard or control were incubated in glass tubes. 100 ~1 of diluted ANT mab (1 : 100) was added except in the blank (added to 100 ~1 of PGT instead of ANT). After mixing, the tubes were covered and left overnight at 4°C. To shorten the experimental time. it could be shown that a minimal incubation time of 2 h at 37°C was enough to reach equilibrium. Dynatech immunoplates were coated with 100 PI/well of LNT, diluted in then saturated with 200 PI/well glycine-PBS buffer, for 1 h at room temperature, PGT for 30 min at room temperature. On the second day of the assay, immunoplates were washed thrice with 200 ~1 of PGT per well, then loaded with the sample mixture: samples of 50 ~1 of the ANT-plasma mixtures were dispensed in triplicate. After 12 min incubation at 37°C. the plates were again washed thrice with PGT. 100 ,uI diluted RAM POX (replaced by 100 ~1 of PGT for the blank) was added, and the plates were incubated for 30 min at 37°C. After 3 washings with PBS (200 ~1 per well. 3 times), 200 ~1 of ABTS in buffer was added. The green color developed slowly as a function of RAM

261 Y

u u 0

yYy A

AMI

0

LNT

0

\

ANT

rc

RAM

-Pox

/

+

substrate

=

ABTS

I

HA

l

absorbance

d

e

Fig. 1. Illustration of the assay procedure. a. ANT is added to plasma sample containing tricyclic antidepressant; the mixture is incubated overnight in glass tubes. b. Mixture is transfered to LNT coated immunoplates. c. After a 12 min incubation at 37OC. plates are washed to remove unbound antibody. d. RAM-Pox is added and allowed to bind to the fixed ANT. e. Substrate is added reacting with the peroxidase, and the growing coloration is measured at 410 nm.

Pox bound to ANT. Absorbance was measured, 45 min after adding procedure described above is illustrated in Fig. 1.

the ABTS. The

Determination of tricyclic antidepressant concentration Calibration curve was plotted as the ratio of mean absorbance for each standard against the amitriptyline concentrations expressed as -log. Unknown samples concentrations were determined by using a linear regression method. Sensitivity and reproducibility were tested on patients plasma samples. Results Specificity The monoclonal antibody directed against nortriptyline was an antibody of IgG, K isotype. Binding studies of the ANT mab, performed by means of the Farr technique with ‘H-DMI showed an affinity constant of 1.447 f 0.177 x lo-’ M for this compound [18]. Inhibition of jH-DMI binding by several tricyclic or unrelated drugs was studied to determine their apparent affinity for the ANT mab. Results of calculated K, are shown in Table I. The amitriptyline group (with carbon atom in position five) bound the ANT mab with slightly higher apparent affinity than the

Drugs

K, (pmol/l)

Amitriptylme Protriptyline Nortriptyline lmipramine Dehlpramine Clomipramine OH-Imipramine OH-Desipramine Maprotlline Oxaprotiline Chlorpromazine Prochlorperazine Phenytciine Chlordiazepoxide Droperidol Alimemazine Levomepromazine Flufenazine Promethazine Diazepam Sulpiride Nitra~epam Haloperidol

0.034 & 0.00x 0.035 * 0.003 0.046 + 0.00X 0.062 + 0.00x 0.070 * 0.00x 0.17Oi 0.050 0.7x0 * 0.0x0 6.X00 i 2.700 7.000 * 1.ooo 124 +31 2.200 +_ 0.400 x.x00* 1.100 20.1 f 4.1 36.4 + 1.3 90.0 * 23.2 > 40 > 40 > 40 > 40 > 40 > 40 > 40 > 40

Plasma therapeutlu concentration (~mol,/l) 0.12 0.30 0.15 0.30

0.05

0.30 2.70 20 3.3

-

6.30

- 5.3 -60 26.7

0.175 0.088 0.09 0.0013-

ICV, 1 +(‘H-DMI)/K,

in which ‘H-DMI of 2 X 10 ’ mol/l.

1.14

1.71

‘7,s

9.4

I20 10

7 0.176 0.23 0.025

Therapeutic and toxic ranges reported in the literature for some constants K,. were calculated from IC,,, usmg the following formula:

K=

T0YlC pla\ma &el ( ~““‘I,‘I)

5.3 0.71 0.13tested

drugs

240 x3

53

1.3

[ZOl. The inhihltion

‘H-DMI

indicates

radiolabeled

desmethylimipramine.

‘H-DMI

was used at the concentration

imipramine group which possess a nitrogen atom in position five. Lateral ring substitutions of imipramine with either chlorine or hydroxyl, decreased the apparent affinity for ANT mab. Among phenothiazines and other psychotropic drugs, binding to antibody was possible for chlorpromazine and prochlorperazine and to a lesser extent for phenytoine. chlordiazepoxide and droperidol.

Culihrution curve The standard curve calculated from 13 different ELBA plates illustrates that 0.095 pmol/l of amitriptyline in serum inhibited 10% of binding of the monoclonal antibody to the coated LNT (Fig. 2). Non-linearity was observed once or twice for

263

-log(i)

pmol/L

Fig. 2. Calibration curve obtained using concentration of the inhibitor: amitriptyline

plasma amitriptyline in the present case.

standards

concentrations lower than 0.088 pmol/l. Therefore, only ,five standards: from 1.4 to 0.088 pmol/l. Precision Repetitive TABLE Precision

ELISA

established

the quality

(mean+sn.

it was decided

control;

results

n = 13). i =

to work with

are shown in Table

II of the ELISA for amitriptyiine

a. Within-day Concentration found with G.C. (pmol/I)

Mean concentration found by ELISA (pmol/I)

SD (pmOi/l)

n

CW)

0.40 0.62 0.89 I.61

1.29 I .49 2.90 4.72

Ct.098 0.21 0.19 0.57

10 7 8 7

7.6 14.0 6.6 12.0

Concentration found with G.C. (pmol/I)

Mean concentration found by ELISA (pmoI/l)

SD

n

W%)

0.87 0.57 0.77 0.47 0.65 0.45 0.65 0.45 0.52

2.30 2.37 1.96 1.25 1.34 1.05 0.71 2.12 1.23

0.13 0.20 0.27 0.11 0.23 0.17 0.08 0.29 0.16

7 8 10 8 9 9 10 8 7

5.4 8.6 13.7 8.7 17.2 16.0 11.7 13.7 13.3

b. Day-to-day

SD,

standard

deviation;

n, number

of samples;

CV, coefficient

( pmOl/i)

of variation,

II.

Within-day precision was estimated by assayin g 7 to IO aliquots from 4 patient\. The coefficient of variation averaged lo’%. Day-to-day precision was cstimatcd hv gathering results concerning 9 patients over 7 to 10 days. Coefficients of variation averaged 12.8%‘. Compurrson

uith gas chromatogruph~~

CC assays were performed (Fernand Widal Hospital. Paris) after extraction. Chromatography was revealed with a selective nitrogen detector [8] adapted to a coiled glass column (4 feet long containing 25% 0 V 101. on Gas-Chrom. Q WHP 100/120 mesh). This technique gave a lower detection limit of 0.10 ~moI/I. Amitriptyline, imipramine and clomipramine were separated through different individual retention times. On the other hand, in the ELISA. the parent drug and its active metabolites were measured at the same time. Over a period of two mth 42 sera collected from patients receiving amitriptyline (Laroxyl) were assayed with both CC and ELISA. The correlation between the two methods was analyzed by linear regression and the following correlation was obtained: r = 0.897: .r = 0.29.~ + 0.15 (Fig. 3). Owing to French medical practice. only a few patients were given imipramine for treatment of depression over the too few samples were collected to establish a same period; as a consequence. correlation with CC for imipramine and its active metabolite desmethylimipramine. However. considering the results obtained for the 5 samples we could gather (data

Antidepressant

Fig. 3. Comparison r = 0.897: n = 42.

of ELISA

concentration

and

Pmol/L

gas chromatography

ELBA

for amitriptyline

asuy:

1‘= 0.29.~ t0.15:

not shown), we predict that attempts to adapt the present method for the assay of imipramine would be successful. On the other hand. consistent with the results of the Farr test, cIomipramine was not recognized well enough by the ANT mab to be measured by means of this ELISA. Discussion We describe here an ELISA using a monodonal antibody directed against &cyclic antidepressants. An ELISA offers several advantages: only small amounts of plasma (100 pi/test) are necessary; no radioactivity is involved, which avoids precautions, specially trained personnel and the necessity of a counter. EL.ISA requires no prior extraction, which shortens the procedure. In this report ELISA was performed over 2 days, with overnight incubation at 4°C but similar results were obtained by a shortened assay: plasma and ANT mab were incubated only 2 h at 37°C. If emergency occurred, results could be returned to medical services within 5 h. Finally an operator can perform at least 50 assays in triplicate per day. ELISA was run with a monoclonal antibody which offers great stability and availability (the same antibody has been produced for nearly 3 yr). The characteristics of the mab have been studied and the epitope recognized established [IS]. Binding properties of the antibody demonstrate that several drugs coultd be measured with this assay. Amitriptyline and imip~amine were measured in patient plasma, nortriptyline and desmethylim~pramine were only tested in vitro since during the period of study no available patients were treated with these drugs. Other compounds like clomipramine could not be measured, since substitutions on the lateral rings of the tricyclic backbone markedly decreased the affinity of the antibody. As shown in Table I, several non-antidepressant drugs might interfere with the assay: a comparison of the Ki obtained by means of the Farr test within the therapeutic ranges reported in the literature [19] shows that only chlorpromazine and phenytoin at clinical doses might interfere if absorbed by patients at the same time. These drugs are not used in common forms of depression, but may be used in association with tricyclic antidepressants, respectively to treat psychotic depression and epilepsy. In contrast, prochlorperazine, droperidol, and benzodiazepines, at therapeutic concentrations, should not interfere with the ELISA. The sensitivity of the method is good: < 0.10 ~mol/l: similar sensitivity is obtained by GC fg]. Higher values of ELISA compared to those determined by GC are the consequence of the binding properties of the antibody. Since drugs and metabolites are recognized with similar affinities, ELISA estimates the total amount of active compounds (amitriptyline and n~rtriptyline), whereas GC provides informations solely on the parent drug level. Individual metabolic pathways leading to a variability in circulating proportions of drug and metabolites could explain some of the apparent discrepancies in the correlation between GC and ELISA. Repeatability and reproducibility seem equivalent to results obtained with some previously described RIA [12,13]. Gas chromato~aphy and HPLC present better precision characteristics: papers report reproducibility coefficients of variation of 8.6% for GC [S]. 10.1% 7% and < 7% for HPLC 122-243. However, these papers also

2hh

mention drawbacks for such performant techniques: extraction procedures and losses. necessity for internal standard. difficulty of column conditioning. pH . solvent and temperature control. volume of samples and duration of the vvhole procedure. Moreover, interference of amitriptyline determination in HPLC with other drugs makes this method less discriminative. Finally, we chose 11) work with effective patients’ samples. and this limited the number of precision tests w/e could run for each patient. Further determinations with an unlimited volume of drug supplemented serum would probably yield lower coefficients of variation. The great rapidity, sensitivity and capacity of this procedure. the reliability of the ANT mab and the moderate cost of reagents and apparatus make this ELISA accessible to all laboratories and suitable for both routine and overdose emergency measurements. Acknowledgments

We thank Dr. F. Baud, Dr. J.M. Scherrmann and Pr. Bourdon (Fernand Widal Hospital, Paris) for their fruitful discussion. The expert assistance in gas chromatography studies of Miss Anne Buisine is gratefully acknowledged. This work was supported by grants from Centre National de la Recherche Scientifique. by a contract ‘Recherche Externe’ from Institut National de la Sante et de la Recherche Medicale, by grants from Association pour le Developpement de la Recherche sur le Cancer, from Ligue de la Recherche contre le Cancer, from University Paris VII and from Ministere de la Recherche et de la Technologie. References 1 Braithwaite RA. Goulding R. Theano G et al. Plasma concentration of Amitriptyline and clinical response. Lancet 1972; June 17: 1297-1300. 2 Kupfer D. Hanin I, Duane GS et al. Amitriptyhne plasma levels and clinical response in primary depression. Clin Pharmacol Ther 1977; 22: 904-911. 3 Hollister LE. Monitoring trlcyclic antidepressant plasma concentrations. J Amer Med Ass 1979; 241: 2530-2533. 4 Scoggins BA. Maguire KP, Norman TR. Burrows CD. Measurement of tricyclic antidepressants. Part II. Applications of methodology. Clin Chem 1980: 26: 805-815. 5 Nicotra MB. Rivera M. Pool JL et al. Tricyclic antidepressant overdose: clinical and pharmacologic observations. Clin Toxicol 1981; 18: 599-613. 6 Rudorfer MV. Cardiovascular changes and plasma drug levels after Amltriptyllne overdose. J Toxicol-Clin Tox~col 1982; 19: 67-78. 7 Scoggins BA. Maguire KP, Norman TR. Burrows CD. Measurement of tricyclic antidepressants. Part I. A review of methodology. Clin Chem 1980: 26: 5-17. X Bailey DN. Jatlow PI. Gas-chromatography analysis for therapeutic concentrations of Amitriptyline and Nortriptyline in plasma. with use of a nitrogen detector. Clin Chem 1976: 22: 777-781. 9 Brunswick DJ. Needelman B, Mendels J. Specific radioimmunoassay of Amitriptyline and Nortrlptyline. Br J Clm Pharm 1979; 7: 343-348. 10 Read GF, Riad-Fahmy D. Walker RF. A radio-immunoassay procedure for Clomlpramine. Postgrad Med J 1977; 3 (Suppl. 4): 110-116. 11 Midha KK. Loo JCK. Charette C et al. Monitoring of therapeutic concentrations of psychotropic drugs in plasma by radioimmunoassays. J Anal Toxicol 1978: 2: 185-192.

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17

18 19 20 21 22

Br J Clin Pharm

1976; 3: 561-565. AI-Bassam MN. O’Sullivan MJ, Gnemmi E et al. Double-antibody enzyme immunoassay for Nortriptyline. Clin Chem 1978; 24: 1590-1594. Hoebeke J, Chamat S, Marullo S et al. Use of the EIA technique in the functional characterisation of anti-drug monoclonal antibodies. in: Avrameas S. ed. lmmunoenzymatic techniques. Amsterdam: Elsevier Publishing Co.. 1983: 3077310. Kamel RS, Landon J, Smith DS. Novel ‘251-labeled Nortriptyline derivatives and their use in liquid-phase or magnetizeable solid-phase second-antibody radioimmunoassays. Clin Chem 1979; 25: 1997-2002. Marullo S, Hoebeke J. Guillet J-G, Strosberg AD. Structural analysis of the epitope recognized by a monoclonal antibody directed against tricyclic antidepressants. J Immunol 1985; 135: 471-477. Veith RC. Perera C. Tricyclic antidepressants. N Engl J Med 1979; 300: 504. Stead AH. Moffat AC. A collection of therapeutic, toxic and fatal blood drug concentrations in man. Human Toxicol 1983; 3: 437-464. Farr RS. A quantitative immunochemicai measure of the primary interaction between *I-BSA and antibody. J Infect Dis 1958; 103: 239. Mellstrom B. Braithwaite R. Ion-pair liquid chromatography of amitriptyline and metabolites in plasma. J Chromatogr 1978; 157: 379-385.

23 Vandemark FL. Adams RF. Schmidt GJ. Liquid-chromatographic procedure for tricyclic drugs their metabolites in plasma. Clin Chem 1978; 24: 87-91. 24 Proelss HF, Lohmann HJ, Miles DG. High-performance liquid-chromatographic simultaneous termination of commonly used tricyclic antidepressants. Clin Chem 1978; 24: 1948-1953.

and de-