Emergency-Room Patients - Semantic Scholar

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doxepin. 6 imiprimine. 7 demoxapam. 8 chlordiaze-. 0.66. 0.59. 0.50 poxide. 9 nortripty- orange line. 10 desmethyl- greenish doxepin. 11 desiprimine. 12.
19. Wide, L., Radioimmunoassays employing immunoabsorbents. Acta Endocrinol., Suppl. 142, 207-221 (1969). 20. Paxton, J. W., Rowell, F. J., and Cree, G. M., Comparison of three radioligands, selenium-75, iodine-125, and tritium, in the radioimmunoassay of methotrexate. Clin. Chem. 24, 1534-1538 (1978). 21. Painter, K., and Vader, C. R., Interference ofiodine-l25ligands in radioimmunoassay: globulin. C/in. Chem.

Evidence

implicating

1254-1255

in plasma. C/in. Pharmacol.

Psychiatry

News, issue no. 2,

3-4 (1979).

23. Hunter,

W. M., Nans, P. W., and Rutherford,

F. J., Preparation

and behaviour of “I-labelled radioligands for phenolic and neutral steroids. In Steroid Immunoassay, E. H. D. Cameron, S. G. Hillier, and K. Griffiths, Eds., Alpha Omega Publishing Ltd., Cardiff, Wales, U.K., 1975, pp 141-152, 204-205.

thyroxine-binding

25, 797-799 (1979). 22. Davis, J. M., Javaid, J. I., Dekirmenjian, H., et al., Comparison between radioimmunoassay and GLC method for haloperidol mea-

CLIN. CHEM. 27/7,

surement

24. Jeste, neuroleptic (1979).

D. V., Rosenblatt, levels in tardive

J. E., Wagner, R. L., et al., High serum dyskinesia? N. EngI. J. Med. 301, 1184

(1981)

Thin-Layer Chromatography, with Fluorescence Detection, of Benzodiazepines and Tricyclic Antidepressants in Serum from

Emergency-Room Patients John M. Meola, Thomas G. Rosano, and Thomas Swift We describe

a rapid, sensitive

procedure

for detecting

benzodiazepines and tricyclic antidepressants in serum. After the compounds are adsorbed on charcoal and eluted with an organic solvent mixture, the drugs are thin-layer chromatographed and detected by their fluorescence. Because it is so fast and easy (turn-around time of about 1 h), the method can be used for emergency toxicology screening. An analysis requires 1.0 mL of serum, and the limit of detection for each of the drugs is about 0.75 mg/L. The procedure is sensitive enough to allow the use of serum instead of urine for the detection of these drugs, because serum has certain advantages over urine as the

sample and better reflects the state of the patient’s toxicity. Addftlonal Keyphrases: drug assay -

methods

.

emergency procedure

The procedure

described

here was developed

as an

adjunct

our laboratory drug-overdose screening procedure (1), which is a rapid gas-chromatographic method, with flame ionization and nitrogen-selective detectors, that facilitates detectionof hypnotic/sedative drugs and also of theophylline and acetaminophen. With the thin-layer chromatographic (TLC) procedure described here, the tricyclic antidepressant and benzodiazepine drugs in the toxic concentrations commonly seen in patients who are brought to the emergency room can be easily and relatively quickly detected. to

These two analytical systems provide us with an adequate system for detecting many of the drugs seen in an emergency room situation, Methods for measuring antidepressants and benzodiazepines by fluorescence (2,3) and spectrophotometry (4) require preparation of fluorescent and other derivatives before analysis. Others require lengthy and difficult liquid chromatography, and therefore are not suited to emergency room determinations. The method proposed here is simple to per-

Albany Medical Center Hospital, Albany, NY 12208. Received Feb. 17, 1981; accepted April 6, 1981. 1254

CLINICAL CHEMISTRY, Vol. 27, No. 7, 1981

relevant

drugs

can

be easily

and

quickly

de-

tected.

Materials and Methods Apparatus Developing

stant-coffee

chamber,

22cm

high, 8cm wide (we use an in-

jar).

“ChromatoVue,” which contains both short (254 nm) and long (365 nm) wavelength ultraviolet lamps (Model C-5

Ultra-Violet Products,

Inc., San Gabriel, CA 91778).

Capillary pipets, 75 mm long, 1.1-1.2 mm i,d. (Krackler Scientific, Inc., Port of Albany, Albany, NY 12202). Chromatographic sprayer (Metroglass, Inc., Boston, MA 02121). Thin-layer chromatographic silica gel plates, 5 X 20 cm,

(cat. Inc.

for the small laboratory

and

form,

no. 4861-620),

Glass traction

Whatman

tubes, 10 X 75 mm and evaporation.

(Kimble

KF6; Krackler no. 73500),

Scientific, used for ex-

Reagents pH 11.0. Dissolve 21 g of sodium carand 420 mg of sodium bicarbonate (NaHCO:,) in 700 mL of water, adjust the pH to 11.0 ± 0.1 with 2 mol/L NaOH or 2 mol/L HC1, and dilute to 1 L with water. Carbonate

bonate

buffer,

(Na,CO,)

Keep refrigerated. Charcoal. “Norit A” neutral, pharmaceutical grade (Amend Drug & Chemical, Irvington, NJ 07111). Extraction solvent, 2 mL of glacial acetic acid/water (66/34 by vol) plus 30 mL of methanol and 70 mL of dichloromethane.

Acetone/dichloromethane/concd. solution (2/3/0.2 by volume). Spray reagent I. Add 200 mg of stannous chloride (SnC12 2H,O) to 50 mL of a 0.6 mol/L solution of HC1, mix, and store at room temperature. Spray reagent II. Add 500mg of stannous chloride to 50 mL of a 120 mL/L solution of perchloricacid. Perchloric acid, 120 mL/L. Dilute 12 mL of a 700 g/L perchloric acid solution to 100 mL with distilled water. Developing

ammonium ‘

solvent.

hydroxide

Table 1. Position and Color under Ultraviolet Light of Spots Produced by Some Drugs Spot no

Color

Drug solvent front

1 2

diazepam

3

desmethyldiazepam

4

amitriptyline

5

doxepin imiprimine

7 8

demoxapam chlordiazepoxide nortripty-

0 72

green

blue orange

0.69

0.66 0.59 0.50

desmethyldoxepin desiprimine protriptyline

11 12

Reference stock from Hoffmann-La tyline, diazepam,

control

greenish brown green brown I. We obtained

Roche, Inc., Nutley, chlordiazepoxide,

working

control.

to an extraction

0,1 mL of reference

0.23 0.14

and des-

stock I was

used for analysis and the methanolic solution was completely evaporated. To the residue was added 1.0 mL of a drug-negative pool, to give a final concentration of 1.0 mg of each drug per liter. This was analyzed as stated under Procedure.

tube

Spotting standards. Each of the drugs was dissolved separately in 10 mL of methanol, and 5 zL of each was spotted on the chromatographic plate with the sample and reference extracts.

Adsorption of drugs. Into appropriately labeled 10 X 75mm glass tubes, place 1.0 mL of sample, 1 mL of carbonate buffer,

and about 4 mg of charcoal. Vortex mix and centrifuge at about 2000 X g for 5 mm. Aspirate and discard as much of the supernate as possible. Add 1.0 mL of distilled water to the extraction tubes. Vortex mix, centrifuge at about 2000 X g for

mm, aspirate, and discard as much of the supernate as possible. Elution of drugs. To the extraction tubes add 2.0 mL of the mixed solvent. Vortex mix vigorously, centrifuge (2000 X g, 1 mm), decant the solvent into a 10 X 75 mm tube, and evaporate at room temperature under air or nitrogen. Chromatography of drugs. Rinse the walls of the 10 X 75 5

tube with two drops of methanol.

length

ultraviolet

light

for the greenish

diazepam

fluores-

cence.

Results and Discussion Table 1 shows the approximate fluorescence characteristics.

Mix gently until the

values of the drugs and

The compounds give very definite, plain fluorescent spots with no background interference. Diazepam fluorescence can be produced by treatment with stannous chloride and hydrochloric acid. This reagent minimizes the background given by the solvent front and so produces a plainly visible diazepam spot. With this solvent system the drugs are separated from their metabolites for ease of identification. Toxic

concentrations

of each of

the drugs, about 1.0 mg/L,

can be easily identified. Gas-liquid or liquid chromatography can be used to confirm the presence of these compounds. Our system allows detection of certain drugs seen often in an emergency room situation, and it is an ideal adjunct to gas chromatography, which easily identifies the sedative hypnotics, the other group of drugs commonly seen in an emer-

gency room situation, No other drug was found to interfere with this system. Drugs investigated for interference were: all the sedative barbiturates and non-barbiturates, anticonvulsants, theophylline, acetaminophen, procainamide, N-acetylprocaina-

mide, caffeine,ethchlorvynol, and salicylate. Because serum is no need for hydrolysis, benzodiazepines

Procedure

mm

pin, and desiprimine-.-any single drug or combination of drugs as required. Allow the solvent to ascend for about 40 mm. Let the plate dry for 5 mm, spray the upper 5cm of the plate with reagent spray I, and immediately check for the presence of diazepam by examining the plate under the long-wave-

sample with those for standards.

diazepam. We obtained protriptyline, imipramine, disiprimine, and nortriptyline from USP-NF Reference Standards, Rockville, MD 20852. Doxepin and desmethyldoxepin were obtained from Pfizer, Inc., Groton, CT. Ten milligrams of each drug was placed in a 100-mL volumetric flask, dissolved in methanol, and diluted to the mark with methanol. Then 1 mL was diluted to 10 mL with methanol to give a concentration of each drug of 10 zg/mL (10

added

de-

desmethyldoxe-

0.28

the following drugs NJ 07110: amitrip-

demoxipam,

nortriptyline,

des-

0 33

methyl

mg/L). Reference

Overspot (i.e., spot on top of one another) diazepam, methyl diazepam, amitriptylene, doxepin, imiprimine,

Spray the remainder of the plate with reagent spray II, warm the plate in an oven at 100-200 #{176}C for 10 mm, and observe the fluorescence of the other drugs under long-wavelength ultraviolet light. Compare the position of the spots, if any, that appear in the

line

10

plate.

moxapam, chloridiazepoxide,

greenish brown green

6

9

1.00 0.88 0.78

green green blue orange

residue dissolves. Using a glass capillary tube, spot the methanolic solution on the chromatographic plate, a few microliters at a time, allowing to dry between applications. The following standards should also be applied on the same

isoniazid, lidocaine, meperidine, is used in this procedure, there as is necessary for detection of

in urine.

References I. Meola, J., Rapid procedure for routine drug overdose screening using GC and the nitrogen-selective and flame ionization detector chromatography. Clin. Chem. Newsletter (published by Perkin-Elmer Corp., Norwalk, CT 06856) 5, 1-3 (1977). 2. Borg, K. 0., and Westerlund, D., Fluorometric determination of non-fluorescent amines by ion-pair extraction. Z. Anal. Chem. 252, 275-278

(1970).

3. de Silva, J., and Strojny, N., Determination of flurazepam and its major biotransformation products in blood and urine by spectrophotofluorometry and spectrophotometry. J. Pharm. Sci. 60, 1303-1314

(1971).

4. Henwood, C. R., Analysis of amitriptyline and its analogues in body tissue. J. Foren. Sci. Soc. 15, 147-150 (1975).

CLINICAL CHEMISTRY, Vol.27,No. 7, 1981

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