The determination of phanquone in biological material by gas-liquid chromatography

THE-DETERMmATIGN OF PIWJ’JQIJGNE IN BIOLOGIC&L GAS-LIQUID CIIRGMATGGRAPHY

P. ti. DEGEN, Rese&ch

S. BRECHBfhILER,

Depa+ent,

PharmnceuticnIs

(ReceivedOctober16th, 1975)

J. SCtiUBLM Didion,

MATERIAL

BY

and W. RIESS

Ciba&e@y

Linziied, &de

(Switzerhmd)

_-

SUMMARY A gas-liquid chromatographic method for the quantitative determination of phanquone is described, based on the formation of a dimethoxime prior to its extraction from biological material. The sensitivity of the procedure is about 15 rig/ml in biological fIuid.

WFRODUCTLON

4,7-Phenanthroline-5,6-dione (phanquone) (I), the active principle of Entobex, marketed by Ciba-Geigy, Basle, Switzerland, camrot be extracted from biological material with the usual organic solvents. By treating the aqueous biological sample with methoxyamine, phanquone can be converted into the 5&dimethoxime (IL), which is a stable, extractable compound that can be isolated by base~specific extraction and subjected to quantitative gas-liquid chromatography (GLC). In the organism, phanquone is partly reduced to 4,7-phenanthroline-5,& dione (V), which shows the same pharmacological activity as the parent compound. As the hydroquinone, under standard reaction conditions used for the derivatization, yields the same derivative as phanquone, the levels determined in biological samples represent the sum of unchanged phanquone and its hydroquinone. In order to correct for losses during derivatization. purification procedures and quantitative gas chromatography, known amounts of JO-methyl4,7-phenanthrohneJ,+iione (C 14 574) (III) are added to the bioIogical sample as an internal standard. MATERIALS AND METHODS Reanents All the chemicals used were of analytical-reagent grade and were tested for purity by carrying out blank runs. Extraction prmedare and derivative formath The procedure is carried out according to the following scheme:

364

-.

To 1-3 ml of pIa&= or u&e: Add 0.5 ml of internalstandard(ca. GO0ng/ml in 0.1 N HCI), 1 ml of buker, pH 3 (67.8 g of citric acid, 25.9 g of NaQH, 17.4 g of KCI in 1000 ml ~of water) and OS ml of 20% (w/v) solution of methoxyamiuehydrochloride.Heat the stoppered tubes in a water-bath at 70” for 2 h.

-4

Add 1.5 ml of 1 N NaOH, 3000 ml of buffer, pH 10 (24.7 g of H~BO~,29.S g of KCI, 14.1 gof NaOH ir?1OKJml of water) and 6 ml of toluene,shake for 15 min on a mechanicalshakerat 200 strolces~mi~. J Centrifi~gefor 5 minutes at 3 rpm (rotor radius = 7 cm). Transfer the organic phase into a dean tube and shake with 5 ml of 1 N H+SO, f?r 15 mill at 20[) strokes/min_ c Aspirate and discardthe organicphase. To the aqueous phaseadd 6.8 ml of 6~NNaOH, 1 ml of buffer, pH 10, and 5 ml of toIuene_Shake for 15 min at 200 strokes/min. Jlknsfer the organic phase into a cleantube and evaporate to dryness under 2 stream of nitrogen at 50”.

,

Add 0.2-O-8 ml of tolueneto the dry residueand i&t

aliquots of 2-5 pl intothe gaschromatograph.

Gas chromatography The instruments used were a Pye ljnicam lModel74, Series 1M, gas chromatograph with a pulsed (250 ,~sec) electron capture detector (-Ni, 10 mCi), an Infotronics

Model CRS 208 integrator and a W+ W Model 1100 recorder. The cohnnns were 5 ft. long, 2 nun I.D. (Pyrex glass) packed with 3 % JXR on Chromosorb G, AW and DMCS, 80-100 mesh. The temperatures used were: column oven, 210”; detector, 300”; injector, 220”. The carrier gas was nitrogen at a ffow-rate of 30 ml/mm. Under these conditions, the retention times of the derivatives of phanquone and the internal standard are about 10 and 13.7 mitt, respectively. The columns were conditioned for 24 h at 280” with a ffow-rate of 15 ml/mm. It should be noted that amounts above 5 ng were not injected as the response ceases to be linear owing to saturation of the detector. Calibration graph

A calibration graph for ihe analysis of phanquone in human plasma was prepared by adding phanquone and internal standard to fresh human plasma and following the above procedure. The internal standard (ITI) (288 ng per sample) and phanquorle were each added as 0.5 ml of a solution in 0.1 N hydrochloric acid. The amounts added ranged from 66 to 500 ng of phanquone per 3-ml sample. The lowest concentration thus determined on this graph was 22 ng/ml. The peak area ratios were plotted against amounts (nanograms per sample) as shown in Fig. 1. Three separate determinations were made for each concentration. The deviation of individual values from . the average did not exceed 10%. The precision was tested by analyzing ten prepared plasma samples with concerm-ations unknown to the analyst. For each concentration, three independent anaIyses were carried out (Table r). Simuhaneousiy, control analyses were performed with concentrations of 80, 300 and 500 ng per sampIe_and the peak area ratios from

GLC

OF PHANQUONE

365

300

200

400

500

ng I sampk

Fig. 1. Calibration graph for human plasma. Sample volume: 3 ml. Internal standard: 288 ng of C 14 574 per sample. Final residue, after anzdyticzl procedure, dissolved in 200 .+I of toluene (t-5 pi inj&ed). Fx = peak area of phanquone-dimethoxime/peak area of C 14 576dimethoxime.

these test analyses were compared with the original calibration graph so as to ensure that the analytical system was stable.

TABLE

I

ANALYSES OF SERUM TO THE ANALYST

SAMPLES

WITri

PHANQUONE

CONC ENTRATIONS

Each sample was analyzed in triplicate.

Sample NO.

1 2 3 4 -5 6 7 8 9 10

Phanquone concentration present ~nglml) 0 135 33 67.6 16.5 268 0 122 243 76

Ptianquone concentration four? (n&d) Mean

S.E.

0 122 33.6 60 18 240 0 142 244 62

0 2.0 0.7 2.9 0.6 9.9 0 7.2 6.7 5.0

Deviation of mean found v&e from concentrarion present (%)

O-0 - 9.6 f 1.8 -11.0 + 9.0 -10.0 0.0 i-16.0 t 0.4 -18.0

UNKNOWN

Plzysical and chemical properties of phnpine a%ne&oxime In order to ascertain the structltres of the dimetho&ne derivatices, 4,7phenanthxoline-5,6done @) and l0-mettryl-4,7-phe~~o~~,~~ione m (Scheme !) were subjected to derivatization in miigram amounts. The GL(= of the productskhowed single peaks and the mass spectra were in accordance with structures 11 and IA? The dimethoxime (II) can he separated by thin-layer chromatography into two isomers @Ia and IIb), which can be identiki by- their NMR spectra. In the symmetrical isomer Ha, the two OCX-& groups (and the protons) are equivalent. Isomer IIb shows no symmetry. Therefore, the two OCPI, groups give rise-to two different signals.

aqueous

solutionP C-Y-

1- @

ENTOBEX

Phanauone

Group

J(Zw

B(PPm) ZLZ

ZZb

OCE~ I-ix H;

4.26 8.71

-4.2 7.75

4.23 7.73

J‘,,,

E&r? H3 &

7-43 8.15

7-42 a;15

7.42 8.14

Ja.3, = l-5 Jcz.3,= 7.5

ZZa = 4.5

4,7-Phenanthroline-5.6-dial (V),

which is known to

be formed

from

phanquone, also leads to the S&dimethoxime (U) when subjected to the reaction with

meeoxyamine (Scheme 1). Pbanquone dimetboxime can be extracted from aqueous soWon with several solvents (toluene, chloroform, ethyl acetate). Although extractioti yields with chloroform and ethyl acetate are higher, toluene was chosen as the solvent as it extracts fewer impurities from biological material that interfere with the GLC determinations. The pH dependence of the partition of phanquone dimethoxime between toiuene and aqueous buffer solutions and plasma is shown in Fig. 2. Purification of extracts from biological material wzs effected by re-extraction into acid (0.1 N sulphuric acid) and back-extraction into toluene at pH 10. ?=recovery (dimethoxime in tokLene phzse) loo

2 .-

4

6 pH (as-

8

10

12

14

ptrase)

depend- oftheextracta~fityof phanq~~oae dimethoxime fromwater (O)and fromplasma (a). Chditioi~~: 1 pg of “C-L&l&d phmquo& dimethoxime, 6 ml of buffersolution(PH O-14) and IOml of ~O~EEE_ E%g. 2. plr

-P. Zi. DEGEN,

368 I

S. BREC&~XXER,

3. SCH&UBLZX,

W. RBZSS

of derivatization 14C-kbeUed phanquone was used for aN expedients

Uptimization

carried out to optimize the reaction conditions. Reaction mixtures were extracted Mh cEIorSo~ at pH 6-7

and aliquots of the organic layer were measured for total radioactivity on a liquid scintillation counter. The percentage of dimethoxime in the reaction product was determined by thin-layer chromatography of the reaction mixture extracts on silica gel with chloroform-acetone (9 :I) as eluent, followed by radiometry of the plates. Treatment of phanquone with methoxyamine hydrochloride iri aqueous solution

A* EcOvery

%

Fig. 3. pH dependence of the formation of mono- and dimethoximcderiv&ives. Conditions: 1 ,ug of phanquone in 1 ml of 0.02 N HCI, 100ml of methoxyamine hydrochloride and 1 ml of buffer solution @H i-12); 2 h at 70”. A, Formation of dimetboxime; 0, form&ion of monomethoxime.

F,

recovery

k&dhodme

in

tduene @aief

metimxylamine

concentration(mg)

F&. 4. Dependence of form&on of pharguone dimetboxime on methoxyamine. hydrochloride concentration.Conditions: t pg of phanquone in 1 ml of 6-02 N-HCI, l-100 mg of methoxyamiine ._ hydroctrlorideznd 1 ml of buff&r CpH 3.0); 2 h at 70”.

363

h

after administration

Fig. 5. Conc~~~trations of unchaaged phanquone in the plasmaof two volunteers afteroral dosesof

50 mg (broken line) and 100mg (solid line) o-f ~W~bex_ Each sample was &yzed

in triplicate.

leads to mixtures of the phanquone monomethoxime and dimethoxime, only the dimethoxime being suitable for the GLC determination. The ratio of monomethoxime to dimethoxime in aqueous reaction mixtures ‘= 3). At pH 2-4, 85 % of the reaction product consists is extremely pH dependent (FI,. of dimethoxime, whereas at pH 7 the monomethoxime is the major component (65 %)The reagent methoxyamine hydrochloride must be present in a very large excess (?O-100 mg per 2 ml of sample, ie., 70,00&100,000fold by weight) in order to obtain the dimethoxime in good yields (Fig. 4). Maximum yields are obtained after 2 h at 70”. Using standard optimal conditions (100 mg of methoxyamine hydrochloride in I ml of water, 1 mi of buffer solution (pH 3), 1 ml of phanquone solution, 2 h at 70”) for 100 ng of phanquone per sample, the yields of dimethoxime are -approximately 80 % from aqueous soWions, approximately 70 % from plasma and 60% from blood. Application of the method to the analysis of human plmmz am2urine

Plasma samples from two healthy human volunteers (A, 49 years, 73 kg; B, 35 years, 65 kg) were analyzeci following the ingestion of A, 50 mg and B, 100 mg of phanquone (as commercial Entobex tabiets). Blood samples were collected at 0, f/2, 1,2,4,6, S and 24 h after ingestion of the dose. Immediately after col.lection, the blood samples were mixed with heparin

P. ET_DEGEN. S. BRECHBmtiR, ~.

370

3. S-UBLiN.

v.

==

and centrifuged. The @asma sampI& were frozen until &&sis. The sampEs Were then processed as described above and chromatographed (Fig. 5).

.

a

lo

8

6

4

:ii

_-co __-___-__-__-,y----4’

2

Fig. 6. Total amount of unchanged phanquone excreted in urine at 0, 5 4,s and 24 h after administration 0: 50 mg (broken line) and 100 mg (solid l&e).

Urine samples were collected during the intervals O-2, 24, 4-S and 8-24 h after administration of the oral dose. The urine sampies were &so kept frozen until required for analysis. The total amount of unchanged phanquone excreted between 0 and 24 h was 12.5% of the dose after the 50-mg dose and 11.2oA of the dose after the NO-mg dose (Fig. 6). ACKNOWLEDGEMENT

We thank Dr. H. Sauter (Central Function Research, Ciba - Geigy) for providing and evaluating NMR Spectra.