Gas Chromatographic Determination of lproclozide in Urine of Psychiatric Treated Patients Roland M. de Sagher,' Andre P. De Leenheer,* and Albert E. Claeys' Facutteit van de Farmaceutische Wetenschappen,Academisch Ziekenhuis, 135, De Pintelaan, 9000 Gent, Belgium

An accurate method for the determination of Iproclozide [l-(p-chlorophenoxyacetyl)-2-isopropyl hydrazine],

Cl-'

' Q

ICH3

-OCH2CONHNH-CH 'C H 3

a potent monoamine oxidase inhibitor drug, in urine samples is important for pharmacokinetic studies and in particular for the assessment of drug therapy. The monitoring of the urinary excretion of Iproclozide during the course of treatment could facilitate adjustment of the maintenance dose and elucidate those pharmacokinetic parameters which explain the wide interindividual variation. Because of the low amounts, 20 to 30 mg range, administered orally, and its extremely rapid metabolism, rather low concentrations of the free compound are excreted in urine. Current interest in our laboratories in the analysis of monoamine oxidase inhibitor drugs have led to the ,development of a specific and sensitive gas chromatographic determination of Iproclozide in urine samples. To our knowledge, no quantitative methods for Iproclozide in biological samples have been reported previously. EXPERIMENTAL Reagents. Iproclozide [l-(p-chlorophenoxyacetyl)-2-isopropylhydrazine] was a generous gift from Coirre (Pharmethic N.V.) and used as such. l-(p-Chlorophenoxyacetyl)-2-(2-butyl)hydrazine was synthesized analogously to D. Libermann and J. C. Denis ( I ) and by the method previously described by us ( 2 ) . All inorganic and organic reagents used were prepared with reagent grade chemicals. High purity nitrogen was used for evaporation of extracts. Collection of U r i n e Samples. Urine samples were collected without preservative from a volunteer (subject 1 and 1') and two psychiatric patients (subjects 2 and 3) to which known amounts of Iproclozide were administered orally. The volunteer was a subject from which urine outputs were collected and analyzed separately after administration of a single dose of Iproclozide (20 and 50 mg for subject 1 and l', respectively). The two psychiatric patients were put under a daily combined drug therapy including Iproclozide (3 x 10 mg and 2 X 10 mg for subject 2 and 3, respectively) and their 24-hr urine outputs were assembled. All urine samples were stored a t -7 "C to the moment the analysis was started. Extraction Procedure for Iproclozide from Urine. T o each 200-ml sample, or the total volume if less is available, of urine in a 1-1. separatory funnel is added 3 ml of 13N N K O H . The solution is extracted once with 400 ml of ether (peroxide free, freshly distilled over hydroquinone, reagent grade). The organic phase is washed three times with 20 ml of 0.1N N K O H and then back-extracted three times with 200 ml of 0.1N HCl. T o the assembled acidic solutions, 10 ml of 13N NH40H are added, and the mixture is extracted three times with 50 ml of chloroform. After addition of the internal standard l-(p-chlorophenoxyacetyl)-2-(2-butyl)hydrazine (60 pg for the volunteer, subject 1 and l', and the patient, subject 2, or 98 pg for patient, subject 3), the chloroform phase is concentrated to a smaller volume in a Rotavapor and finally under a nitrogen stream evaporated in silanized conical centrifuge tubes of 15-ml capacity until dryness. The residue obtained is dissolved with the aid of a Vortex-type mixer in 100 pl of chloroform. Laboratorium voor Analytische Chemie. Laboratoria voor Medische Biochemie en voor Klinische Analyse. 1144

ANALYTICAL CHEMISTRY, VOL. 47, NO. 7, JUNE 1975

Instrumental Conditions. A Varian Aerograph 2800 gas chromatograph equipped with dual flame ionization detectors was fitted with spiral silanized glass columns packed with 1% Versamid930 on 80-100 mesh Gas Chrom Q (3-mlength, 2-mm i.d.), 5% XE60 on 80-100 mesh Gas Chrom Q (2.5-m length, 2-mm i.d.) and 5% OV-225 on 80-100 mesh Gas Chrom Q (2.5-m length, 2-mm i.d.). All columns were conditioned for at least 12 hours at 220 OC with a carrier gas (nitrogen) flow of about 5 ml min-' and then for a few hours a t operating conditions. Analyses were carried out under isothermal conditions a t oven temperatures of 160, 170, 180, or 190 "C with a few degrees higher for the injector and detector blocks (FID). Nitrogen was used as a carrier gas a t linear velocities PO, measured with methane, covering the range 9.4 to 14.3 cm sec-l but kept constant within each quantitative series. The hydrogen and air flow rates were adjusted as to give optimum sensitivity and good stability: hydrogen, 24 to 38 ml min-' and air, 250 to 550 ml min-'. The detectors were as usually connected to the electrometer and via an Infotronics instrument CRS-104 for electronic integration of peak areas to a Varian Aerograph A25 recorder, 0-1 mV range. The attenuation settings employed were 2 X A mV-' for the electrometer whereas the electronic integrator input/output attenuator was used at X 5, x 10, or x 20. All injections were made with a 10-pl Hamilton 701 N syringe on top of columns using an injection volume of approximately 1 pl. Calculations. Calibration factors k, were determined by measuring corresponding peak areas on chromatograms from the calibration series consisting of five different solutions of equal weight ratio increments:

hi = AIc~i.,./Ai.s.~~~c where AI, and A , and wtCand w , are peak areas and weights of Iproclozide (IC)and internal standard (i.s.), respectively. For each calibration series, the mean calibration factor R was calculated using four or five measurements. Results for urine samples C, (pg/100 ml) were found by the equation:

cu

100 -

0.91 L:

--+

1 A' A

zui.s.

where 100/0.91 u is the conversion factor for an overall recovery of 91% and an extracted volume u (ml) of urine; k is the mean calibration factor; A ' I ~and A'i.s. are peak areas of Iproclozide as present in the chromatogram of the urine extract and the internal standard added to the chloroform phase, respectively; and w ~ .is~ the . weight of the internal standard added to the chloroform extract prior to evaporation. The urinary excretion E , (%) or percent of administered dose per 24 hours, recovered as unchanged drug, is calculated as follows:

E, = Cu V / D where C, (pg/lOO ml) is the urinary concentration of Iproclozide; V (ml) is the total 24-hours urinary output and D (pg) is the amount of Iproclozide administered orally in a 24-hour period.

RESULTS AND DISCUSSION Gas chromatography operating conditions, net retention times ( t ~of) Iproclozide (IC) and the internal standard (is.), relative retentions r and calibration data (K) are given in Table I. The three liquid phases Versamid-930, XE-60, and OV-225 permit chromatography of Iproclozide and the internal standard without any prior derivatization. Because of its close structural relationship, the 2-butyl analog is undoubtedly the internal standard for preference. Extraction of biological samples with organic solvents a t

Table 1. G a s Chromatography Conditions of Iproclozide (IC)a n d t h e Internal Standard ( i s . ) Oven tem-

Linear v e -

OC

1%Versamid-930

160 170 180 190

5%XE-60

5% OV- 225

sec.

locity o;

peramre, Column system

Retention time t R,

c m sec-1

Mean caliRelative re-

IC

i. s.

9.4 9.4 9.4 9.4

2253 1379 942 634

3404 2045 1371 903

0.663 0.675 0.688 0.702

160 170 180 190 190

14.3 14.3 14.3 14.3 6.8

1397 a84 592 404 9oa

1989 1237 813 547 1232

0.702 0.715 0.729 0.737 0.737

180

10.4

1288

1801

0.714

100

Table 11. Recovery of H u m a n Urine Samples Spiked with Iproclozide

tention

T

bration factor F

1 .oo

0.99 0.99

1 .oo

-

Y

L M

L

-

z

Amount of Ipro-

E

cluzide added to

Amount of Ipm-

2 0 0 rnl of urine,

clozide recovered,

Recovery,

ug

ug

%

20 40 60 80 100

17.7 36.5 54 .o 73.7 91 .o

89 91 90 92 91

Y E

-

O 0 E

u, D

c

-

Table 111. Relative Retentions r of Iproclozide a n d Interfering Compounds as Present i n Urine Extracts Oven t e m -

Relative retention r

peranue,

Interfering

Column system

OC

lproclozide

compounds

1% Versamid

160 170 160 170 1 a0 190

0.662 0.675 0.702 0.715 0.729 0.737

0.63 5 0.664 0.716 0.739 0.760 0.773

5 % XE-60

defined pH values is often carried out as the first step for isolation and extraction of drugs. To develop such a method, the partition coefficients of Iproclozide between chloroform or ether and an aqueous basic or acidic phase were measured (partition coefficient, extractant/extracted phase: 70, CHCls/aq NaOH soln; 2.0, ether/aq NaOH soln; 230, CHCls/aq NH40H soln; 12, ether/aq NH40H soln; 0.25, 0.1N HCl/CHC13 and 4.0, 0 . 1 N HCl/ether). These results indicate that Iproclozide is the better extracted from an aqueous NH40H phase with ether from which it is easily back-extracted into 0.1N HC1. The loss of compound during the first extraction step with ether is 4.0% whereas the back-extraction into 0.1 N HC1 is 96.3% efficient. Transfer to the organic phase might then be performed after adding an excess of 13N "*OH and using chloroform as the extractant. Five 200-ml urine samples spiked with 20,40,60,80, and 100 pg (amounts in the range anticipated to be present after minimal dosage) of Iproclozide, respectively, were also examined under conditions described. Results obtained on the 5% OV-225 column operated at 180 O C are presented in Table I1 and show a recovery relative to the internal standard of 90.6 f 1.1%, a figure

TIME IN MiNUTES

Figure 1. Gas chromatogram of a urine extract from the volunteer, subject 1 The column was 5 % OV-225; quantitative determination of lproclozide (IC) with the 2-butyl analog as internal standard (is.)

that correlates excellently with the theoretical value of 91% calculated from partition data. The sample workup probably eliminates many potential interferences. Removal of these is definitely aided by switching the organic solvent during the extraction procedure. In addition to this a good chromatographic system should separate completely the compound to be determined and its internal standard from biogenic products coextracted. However, as was noticed in multiple analyses of blank urine samples, all column types when used at 180 "C were not equivalent nor successful in this respect. The 1% Versamid-930 column seemed unsuitable for analysis of small amounts of Iproclozide because the presence of an unidentified compound with the same relative retention as Iptoclozide itself. Decreasing the oven temperature below 160 "C should well enable identification of Iproclozide. The 5% XE-60 column allowed clearcut qualitative analysis but also proved unappropriate for a sensitive quantitative determination (falsely elevated results equivalent to about 3 pg of Iproclozide due to caffeine as an interfering product ANALYTICAL CHEMISTRY, VOL. 47, NO. 7, JUNE 1975

1145

Table IV. Quantitative Results of Gas Chromatographic Determinations of Iproclozide in Urine Samples Urinary excretion

C, (ug/100 ml)

Sample

E , (%) Subject

Time, hr"

Volume, m e

1% Versamid-930

5% XE-60

[5% OV-225 1

5% OV-225

1

1 2 3 4

1.5 5 .O 7.5 12.5

86 178 119 146

17.1 27.5 5.6

22.6 28.7 12.8 8.7

14.4 25.3 2.3 2.6

0.06 0.23 0.01 0.02

1'

1 2 3 4

2.5 6.5 12.5 22.5

108 209 211 305

94.5 17.4

99.1 17.1 7.1 1.9

105.4 14.9

0.23 0.06 0.03 0.01

2

1 2 3 4 5 6

16 .O 17.5 5 .O 6.8 12.9 6.6

13.6 17.3 4 .O 5.4 12.8 4.7

13 .O 15.9 3.7 6.9 13 .O 4.8

0.45 0.56 0.10 0.16 0.37 0.15

3

1 11.8 0.35 2 1.8 0.08' 3 17 .O 0.41 4 2.5 0.19 a Time in hours after oral intake of Iproclozide. Volume in ml of each urine sample voided. Estimated value too low, incomplete sample.

10

20

30 TIME I N HOURS

Cumulative amount excreted (pg) of lproclozide in urine as a function of time (hr) for a single dose administration of 20 mg (0) and 50 mg ( 0 )respectively Flgure 3.

J

0

Figure 2.

IO

20

10

40 TIME IN MINUTES

Gas chromatogram of a urine extract from patient, subject

3

The column was 5 % OV-225; quantitative determination of lproclozide (IC) with the 2-butyl analog as internal standard ( i s . ) in the extracts). Less overlapping was obtained a t higher oven temperatures (Table 111) and using a lower carrier gas flow rate (6.8 cm sec-l instead of 14.3 cm sec-I). Finally, the 5% OV-225 column was certainly most acceptable: only a very small bump, equivalent to an absolute amount of 0.4 to 0.5 pg of Iproclozide, with a relative retention r 0.760 was detected but did not interfere seriously with results obtained. Furthermore the region where the internal standard elutes is essentially free from interferences. In order to verify the applicability of this analysis to experimental subjects, Iproclozide was taken orally in single 1146

ANALYTICAL CHEMISTRY, VOL. 47, NO. 7, JUNE 1975

doses of 20 and 50 mg by a volunteer and his successive urine outputs were collected and analyzed separately. Furthermore, the 24-hour urine outputs of two psychiatric patients under combined known drug therapy (orally, 2 X 10 or 3 X 10 mg of Iproclozide) were also taken through the entire procedure. Representative chromatograms are shown in Figures 1 and 2. The cumulative amount of Iproclozide excreted in urine of the volunteer as a function of time is presented in Figure 3. For the single dose administrations, the Iproclozide urinary excretion falls within a few hours and then decreases much more slowly. It is noteworthy that, in this experiment, samples were collected as long as 52.5 and 36 hours, respectively, after drug administration and Iproclozide could still be detected after 12.5 and 22.5 hours for 20- and 50-mg intake, respectively. Under conditions mentioned, back-ground interference did not present a problem at concentrations of Iproclozide as low as 0.02 pg per ml of urine. Quantitative results of gas chro-

matographic determinations of Iproclozide in urine samples Of the two psychiatric patients are shown in IV' The percent urinary excretion of Iproclozide in 24-hour Periods varies from 0.15 to 0.45% (as measured on the most selective 5% OV-225 column) of the dose of active substance orally administered each day. It also must be emphasized that no interferences from the other drugs or their metabolites were experienced in these analyses as was proved by mass spectrometric verification of peaks eluted and measured.

LITERATURE CITED (1) D. Libermann and J. C. Denis, Bull, Soc. Chim. Fr., 2, 1952 (1961). (2) R. M. de Sagher, A. P. De Leenheer, and A. E. Claeys, J. Chromatogr. Sci., in press (1975).

RECEIVEDfor review December 12, 1974. Accepted February 3, 1975. The authors thank the N.F.W.O. for giving a bursary (aspirant) to one of us and the F.G.W.O. for support through Grant 20210.

Simple Method for the Determination of Uranium in Soils by Two Stage Ion Exchange Talk Baltakmens Department of Health, National Radiation Laboratory, 108 Victoria Street, Christchurch, New Zealand

In the radiochemical analysis of environmental samples, the determination of uranium is frequently a problem due to its complex chemistry. This is particularly true for soils, where the low concentration of U, presence of other radionuclides, and interference by a number of stable elements makes the extraction and separation of U quite difficult. Numerous methods for the determination of U in trace quantities (ppm range) have been developed, (1-3), but most are complicated and time consuming, requiring special reagents and apparatus. Other methods are based on * very sophisticated techniques of analysis, e.g., neutron activation combined with X-ray spectroscopy ( 4 ) also requiring special equipment, while simpler methods do not always give complete separation of U from other radionuclides (5). This paper presents a simple method for determining U in soils by a two stage ion exchange separation followed by alpha counting. The procedure is simple and effective, gives >95% recovery of U, eliminates interference by other natural radionuclides, requires only ordinary laboratory reagents and ion exchange resin, does not need elaborate instrumentation, and the analysis can be completed in a reasonably short time. Up to the final step, where the U source is prepared for alpha counting, the chemical procedure does not involve any precipitation steps, thus avoiding possible loss of U due to solubility or complex ion formation. EXPERIMENTAL Procedure. Dry the soil to be analyzed a t about 105 "C for 1-2 hours, let cool, crush, and sieve a portion of it through a 150-mesh sieve. Weigh out 1.0 g of this portion and ash it in a muffle furnace a t 600 "C for about 1 hour to destroy organic matter and facilitate the breaking down of the soil minerals. (The ashing can also be done in a nickel crucible over a Meker burner). After cooling, mix the sample with 3 ml of concd nitric acid and 10 ml of 40% hydrofluoric acid in a small platinum basin and evaporate to dryness over moderate heat. (Alternatively,the mixture can be evaporated in a polythene beaker over low heat, e.g., on a water bath). Dissolve the dry sample by boiling gently for a few minutes with 8N hydrochloric acid, let the solution cool and filter with suction through a suitable filter (e.g., a 4.25-cm diam Whatman No. 52 filter paper in a Buchner funnel). Some soils leave an insoluble residue a t this step, but in most cases this constitutes only 1-2% of the total weight of the sample and contains no detectable alpha activity. Pass the filtrate through an 0.8-cm diam anion exchange column

containing 8 ml of de-Acidite FF I P SRA 66 52-100 mesh (or equivalent) resin, which has been pretreated with 15 ml of 8N HC1; allow a flow rate of about 1-1.5 ml/min. Wash the column with 15 ml of 8N HC1, followed by 25 ml of 0.5N HCl. Evaporate the 0.5N HCl eluate to dryness over low heat, then dissolve the residue by warming with 5 ml of 2N sulfuric acid. Cool the solution, dilute to 100 ml with demineralized water and pass through an identical column as above, this time pretreated with 15 ml of 0.1N HzS04. Wash the column with 50 ml of 0.1N H2S04 and then pass through 50 ml of 0.5N HC1. Discard the first 15 ml of the 0.5N HCl eluate and collect the remainder. Add 1 ml of yttrium carrier solution, equivalent to 2.8 mg of yttrium hydroxide, heat to boiling, remove from heat and co-precipitate U on Y(OH)3 by the addition of carbonate-free ammonium hydroxide. Cool, filter the precipitate on a tared 2.5-cm GFA glass fiber filter, dry, weigh, cover with zinc sulfide on Mylar and alpha count in a scintillation counter. Determine the counting efficiency for a suitable range of precipitate weights with a U standard.

DISCUSSION AND RESULTS Of the natural radioactive elements present in soil, U, Pa, Bi, Po are absorbed on the first column (in 8 N HCl); Th, Ra, Ac, P b are not absorbed. Of the major stable elements only Fe(II1) is absorbed; Al, Ca, Mg are not absorbed. Elution with 0.5N HCl removes U, Pa, and Fe; Bi and Po remain on the column. U and P a are absorbed by the second column (in 0.1N HzS04) together with a small amount of Fe. Washing with 50 ml of 0.1N H2S04removes most of the absorbed Fe; traces of it may appear in the first 15 ml of the 0.5N HCl eluate. The 15-50 ml fraction of this eluate contains all the U. In calculating the U content of the sample from its alpha activity, radioactive equilibrium between 238U and 234U is assumed, which is the case for most rocks and soils (6). In cases where it is suspected that this equilibrium may not be established, the exact proportion of each of these two U isotopes can be found by beta counting the Y(OH)3 source after a suitable interval to allow for the ingrowth of the beta emitting 238Udaughter a34Th(T1/2 = 24.1 days). The beta activity which is then registered on the beta counter is that of the short lived daughter 234mPa( T I / *= 1.18 min) of 234Thin radioactive equilibrium with it; since the betas from 234Thitself are of too low energies to make a significant contribution to the counting rate. Thus, the beta ANALYTICAL CHEMISTRY, VOL. 47, NO. 7, JUNE 1975

1147