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Cfin ico Chimica Acta, 17.5 (1988) 175-182

Elsevier

CCA 04196

more simple, rapid and sensitive ~uo~~etri~

method for the det~~ati~~ of isoniazid

and acetylisoniazid in serum.

App~cation for acetylator ~~enot~i~~

Pinelopi C. Ioannou

Laboratory ofAna ~ticalChem tiy, Chemistry Department, Universi@of thens, Athens (Greece)

(Received 24 September 1987; revision received 26 3jebrwq 1988;

accepted after revision 22 March 1988)

Ke,v words: Isoniazid; Acetylisoaiazid; Fkorescent analysis; Fluorescent ch~ate-ph~ot~~~ test

A simple, rapid and sensitive fluorimetric method for isoniazid determination in

serum is described. The method is based on the reaction of isoniazid with 2&y-

boxy-l-naph~~dehyde in acidic medium in the presence of excess of scandium.

Wit-an precision (Cv) was lS%, 1.0% and 1.2% at mean isoniazid concentration

in serum of 0.244, 1.94, and 25.9 mg/l respectively (n = 10); between-run precision

(CV) was 3.055, 2.6%, and 1.9% at mean isoniazid concentration of 0.265, 1.93, and

26.2 mg/l respectively (n = 10). The linearity of the method extended over the range

of O-300 mg isonia.zid/l serum. The detection limit (defined as three times the SD of

the mean blank) for the method is 0.008 mgfl of serum. Samples from 80

tuberculous patients treated with isoniazid were analysed by the proposed method

(y) and by the modified spectrofluorimetric method of Miceli et al (x). Linear

regression analysis of the results yielded the equation y = 0.98x + 0.05 fr = 0.986,

Sxy = 0.22).

Isoniazid (isonicotinic acid hydrazide, INK) is considered to be the most effective

of the commonly administered antituberculosis drugs. INH, like sulfamethazine, is

Correspondence to: P.C. Ioatxnou, Laboratory of Analytical Chemistry, Chemistry Department, Wniver-

sity of Athens, 104 Solonos St., Athens 10680, Greece.

~9-~981/88/ 0~.50 6 1988 Elsevier Science Publishers B.V. (Biomedical Division)

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76

acetylated and inactivated in the liver by the acetyl-transferase enzyme [1,2]: the

rate of acetylation is genetically controlled.

Several methods have been used to measure INH and acetylisoniazid (AcINH) in

serum, including calorimetry [3], spectrophotometry [4-61, fluorescence analysis

[4,7], and high performance liquid chromatography (HPLC) [8,9]. Calorimetric and

spectrophotometric methods are time-consuming, not sensitive enough to measure

the relatively lower concentrations of INH and AcINH in serum, and are not free

from interferences. On the other hand, analysis by HPLC is more specific, but has

the laborious steps of separation from biological material and derivatisation prior to

the injection of the sample into the chromatograph to improve the sensitivity of the

method. Fluorimetric methods for INH determination are the most sensitive,

especially the method of Scott and Wright [7], which was improved by Miceli et al

[lo], and was modified and adapted for AcINH determination by Olson et al [ll].

However, this method too is time-consuming (the procedure for INH determination

takes more than an hour), and laborious for use in clinical practice. For clinical

purposes a simple, rapid and sensitive method for INH and AcINH determination

would be advantageous.

Recently, I reported a new fluorimetric method for the determination of INH in

aqueous solutions [12]. The method was based on the reaction of INH with

2-hydroxy-l-naphthaldehyde (HNA) in acidic medium in the presence of scandium

excess with the consequent formation of a strongly fluorescent complex (A, = 430

nm, A,,= 510 nm) between the hydrazone formed and scandium in weakly acidic

medium. For INH measurement two procedures were proposed, a kinetic procedure

(reaction time 1 min), which was less time-consuming with fewer manipulation

steps, and an equilibrium one (reaction time 10 min), which allowed the avoidance

of the precise timing control.

The present study represents an application of this method for the determination

of INH in serum using the equilibrium procedure. To avoid the interference from

serum components, removal of proteins by treatment with acetonitrile is required.

Concentration values are calculated against an aqueous standard curve, which is

similar in slope to curves constructed with data on serum supemates. The method is

adapted for the determination of AcINH in serum after gentle acidic hydrolysis to

INH, and for acetylator phenotyping by estimating the ratio of AcINH (expressed

as INH) to the apparent INH.

Materials and methods

Instrumentation

A Model 512 fluorescence spectrophotometer (Perkin-Elmer Corp., Norwalk, CT

USA) with a 150 W xenon lamp, equipped with a magnetic stirrer under the cell

holder, was used. The instrument settings were as follows: ratio mode, excitation

wavelength 430 nm, with a band width of 20 run; emission wavelength 510 run, with

a band width of 20 mn. A constant temperature of 25.0 o C in the 1 Ooo-cm sample

cell was maintained with a thermostated water bath. The sonicator used was from

Struers Co., Model Metason C., H 50-60. Finpipette microsyringes for transfer of

small sample and reagent volumes were used.

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177

Reagent s

All solutions were prepared in deionised, distilled water from reagent-grade

materials, unless otherwise stated.

HNA solut i on, 10.0 mmoE/I , i n acet onit ri le.

Prepared from HNA obtained from

Fluka. HNA solution is stable at room temperature for several weeks.

Scandium sol~io~,

20.0

rnrn~~~l.

Prepared from scandium oxide specpure,

Johnson, Mattey and Co.) according to [12]. A 10.0 mmol,/l working solution with

pH 1.1 prepared by appropriate dilution with water and few drops of saturated

sodium hydroxide solution.

Stock buffer solution

0.1 mol/l sodium acetate, 10.0 g/l hydroxylammonium

chloride), pH 6.3. Working buffer solution prepared by mixing one volume of the

stock buffer solution with one volume of acetonitrile.

Standard INH

solution

1000 mg/l,

in water

Prepared from INH obtained

from Sigma. Stored at 4C this solution is stable for several weeks. Working

solutions were prepared by appropriate dilution daily.

St andard AcIN H solut ion, I ~.0 mg/l.

Prepared according to the method of

Eidus at al [13].

Sodium hydroxide, I mol/l.

Hydrochlori c aci d 1 mol /l .

Pool ed sera .

A normal serum pool was prepared from blood drawn from healthy

volunteers and stored in 18 x 118 mm polypropylene tubes Kartell, Binasco, MI,

USA) at 4OC.

Tuberculous patients samples.

The

patients were given orally a single dose of

300 mg INH, and blood specimens were obtained 4 h later. Blood was allowed to

clot and was centrifuged for 10 min at 1500 X g. Serum specimens for INH

dete~ation can be stored at 4 o C for several weeks without deprote~tion. For

AcINH determination the removal of proteins within 24 h after the serum were

collected is essential because of the instability of AcINH in serum [ll].

Metho

Sampl e preparat i on.

To remove protein, 100 pl of serum was mixed with 200 ~1

of acetonitrile in a polypropylene test tube, stirred for 60 s on a vortex-type mixer,

and centrifuged for 5 min at 1500

x g.

INH measurement.

100 ~1 of the serum supernate was transferred to a test tube,

100 ~1 of scandium working solution added along with 50 ~1 of HNA solution. The

mixture was stirred for 10 s and agitated vigorously in an ultrasonic bath for 10 min.

2.00 ml of the working buffer solution was added, the mixture stirred and the

fluorescence intensity measured. A standard curve based on aqueous INH standards

was prepared by mixing one volume of the INH standard solutions with two

volumes of acetonitrile. The procedure for INH was followed, and the specimen

con~ntration was calculated from this curve.

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178

Total hydrazides (I NH plus AcI NH ) measurement.

100 ~1 of serum supemate

was transferred to a test tube, 20 pl of 1.0 mol/l hydrochloric acid added, the tube

capped (cap must be pierced), and the mixture heated in a water bath at 80C for

an hour. After cooling the mixture, 20 ~1 of 1.0 mol/l of sodium hydroxide was

added and the measurement performed as above.

Ana~tica~-recoue~ experiments.

200

~1 of pooled serum were pipetted to each of

twelve polypropylene tubes, 20 ~1 of aqueous INH standards were added and mixed

thoroughly. After protein precipitation, INH measurements were performed three-

fold for each sample.

Results and discussion

The optimum experimental conditions of the HNAINH-hydrazone and

HNAINH-SC chelate formation (optimum pH, reagents concentration, standing

time, ~terfer~ces, etc.) have been studied [12]. According to the method described,

the fluorescence intensity of the HNAINH-SC chelate, which was measured after

the buffer addition, decreased by 15% during the first 2 min and then remained

stable. In the present study the nature of this phenomenon was examined in order to

eliminate this decrease and to simplify the measurements. It was found that this

decrease was due to the presence of Fe3+ traces in solutions, which caused the

quenching effect. Because of the non-interference from the Fe2+, the addition of

some reducing agents, such as ascorbic acid and hydroxylammonium chloride, was

examined. The best results (no decrease in fluorescence intensity) were obtained

after the addition of hydroxyl~o~~ chloride to the acetate buffer at a

~on~n~ation of 10.0 g/l and pH 6.3 in the final buffer solution. It was also found

that the pH of the a~e~t~hydroxyl~o~~ solution remained stable after

acetonitrile addition.

In order to choose the better technique for the measurements, I calculated the

mean slope for aqueous standard curves (relative fluorescence intensity,

F vs

INH

concentration, mg/l), which was found to be 6.44

F .

(mg/l)- SD = 0.03, n = S),

and the mean slope for standard addition curves with serum supemates after protein

precipitation, which was found to be 6.38

F .

(mg/l)-1 SD = 0.26, n = 20). The

mean slope in both cases correlated well showing no interference from the serum

matrix, so INH ~n~ntration values were calculated against an aqueous standard

curve. Linearity of the method extended from O-300 mg/l using the recommended

procedure, and can be increased to the concentration above 300 mg/l by reducing

the supemate volume taken for the measurement. The detection limit (defined as

three times the SD of the mean blank) for the method is 0.008 mg/l of serum, or

0.005 mg/l using 200 ~1 of the serum supemate for the measurement. To determine

the within-run and between-run precision, serum pools containing different INH

concentration were measured. For within-run precision assessment, three samples,

after protein removing, were measured 10 times each. For between-run precision, I

performed protein precipitation 10 times for each of the samples and measured the

INH con~ntration in triplicate for each. Table I shows the precision results for

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179

TABLE I

Precision data

a(mg/l)

sD (mg/l)

cv @)

Within-run

Between-nm

0.244 1.94 25.9

0.265 1.93

26.2

0.004 0.02 0.3

0.008 0.05

0.5

1.6 1.0 1.2

3.0 2.6

1.9

n = 10, throughout.

serum samples of different INH concentration. The CV values obtained confirm the

high reproducibi~ty of the proposed method. Analytical recovery experiments on

pooled serum samples, which were performed for the method evaluation, are shown

in Table II. Recovery ranged from 90.0-105.28 (mean 98.7%) for three INH

concentration ranges and indicated that using acetonitrile for protein precipitation,

INH could be obtained practically quantitatively in serum supernate. Comparison

data for INH dete~nation on 80 patients samples, as assayed by the proposed

and by Miceli methods, yielded the following linear regression equation: y = a + bx

(a = 0.05, SD = 0.04; b = 0.98, SD = 0.02); the standard error of estimate = 0.22;

r = 0.986 (x-axis, reference method; y-axis, proposed method). The INH concentra-

tion in plasma varied from 0.20-5.9 mg/l. To adapt the proposed method for

AcINH determination and afterwards for phenotyping of patients, the hydrolysis of

AcINH on pooled serum was studied, and recovery experiments on pooled serum

spiked with AcINH and INH were performed. As it was found, the hydrolysis of

AcINH to INH in serum supemate in the presence of hydrochloric acid solution is

completed in an hour at 80C. Recovery for INH measured in aliquot A, total

TABLE II

Analytical recovery data of INH added to serum

INH added (mg,/l) INH measured (mg/l)

0.250

0.242

0.500

0.464

0.750 0.789

1.00

1.01

2.50

2.30

5.00

5.18

7.50

7.48

10.0

10.1

20.0

18.0

30.0

31.5

50.0

46.8

60.0

62.0

INH recovery ( )

96.8

92.8

105.2

101.0

Mean 98.9

92.0

103.6

99.7

101.0

Mean 99.1

90.0

105.0

93.6

103.4

Mean 98.0

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TABLE III

Analytical recovery data of INH and AcINH expressed as INH) added to serum

INH mg/l) a AcINH mg,/l) b

Total mg/l)

Added Measured Recovery Added Measured Recovery

Added Measured Recovery

2.0 2.0

100.0 3.0 2.8 93.3 5.0 4.8

96.0

4.0

3.8 95.0 6.0

6.2 103.3 10.0

10.0 100.0

6.0

5.9 98.3 9.0

8.4 93.3 15.0

14.3 95.3

8.0

8.1 101.3 12.0

11.2 93.3 20.0

19.3

96.5

Mean 98.7

Mean 95.8 Mean 97.0

Measured in aliquot A.

b Calculated as total INH in aliquot B-MH in aliquot A).

Measured in aliquot B.

hydrazides measured in aliquot B, and AcINH calculated as (INH in aliquot B-INH

in aliquot A) ranged from 93.3-103.38 (Table III). For the classification of the

patients into slow or fast inactivators, the ratio of AcINH (expressed as INH) to the

apparent INH was estimated:

AcINH-Index =

(total INH in aliquot B-INH in aliquot A) (mg/l)

(INH in aliquot A) (mg/l)

All the 80 samples were phenotyped by the proposed method, and according to

[8,14] 52 of them (65%) with an average AcINH-index of 0.71 (0.10-1.30) were

classified as slow and 28 of them (35%) with an average AcINH-index of 3.10

(1.70-11.30) as fast inactivators. Several drugs, such as rimfampicin, p-aminosali-

cylic acid, theophylline, sulfamerazine, sulfanilbenzamide, that may be used in

conjuction with INH, were tested to see if they interfere in the assay. When toxic

concentrations of each drug listed were added to standards of INH, no quenching,

interference, or increase in fluorescence intensity was found.

The proposed method is more simple and less laborious than the other fluorim-

etric methods, very sensitive, and permits the use of as little as 100 ~1 of serum for

both INH and AcINH measurements. The deproteinization with acetonitrile is

found to be more effective than by other methods, gives excellent recoveries and

eliminates the effect of serum matrix, especially metal ions, such as Fe3+ and Cu2+,

which cause quenching effect. The method is suitable for routine use in clinical

laboratories, and can be automated (kinetic procedure) using deproteinized samples

with various analysers, such as flow injection, air-segmented continuous flow, or

discrete analysers with a fluorimetric detection.

cknowledgements

I thank Ms. Ana~ostopo~ou, Mr. Marinis and his colleagues at

ology Laboratory, Hospital Sotiria for Thorax Diseases {Athens,

supplying the patients samples.

the Microbi-

Greece) for

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